High eicosapentaenoic acid oils from improved optimized strains of yarrowia lipolytica

ABSTRACT

Described are engineered strains of the oleaginous yeast  Yarrowia lipolytica  capable of producing an oil comprising greater than 50 weight percent of eicosapentaenoic acid [“EPA”], an ω-3 polyunsaturated fatty acid, measured as a weight percent of total fatty acids [“% TFAs”] and having a ratio of at least 3.1 of EPA % TFAs, to linoleic acid, measured as % TFAs. These strains over-express at least one Δ9 elongase/Δ8 desaturase multizyme, in addition to other heterologous Δ9 elongases, Δ8 desaturases, Δ5 desaturases, Δ17 desaturases, Δ12 desaturases, C 16/18  elongases, and optionally over-express diacylglycerol cholinephosphotransferases, malonyl CoA synthetases and/or acyl-CoA lysophospholipid acyltransferases. The strains possess at least one peroxisome biogenesis factor protein knockout. Methods for producing EPA within said host cells, oils obtained from the cells, and products therefrom are claimed.

This application claims the benefit of U.S. Provisional Applications No.61/187,366, No. 61/187,368 and No. 61/187,359, each filed Jun. 16, 2009and each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to an engineered strain of the oleaginous yeastYarrowia lipolytica that is capable of efficiently producingeicosapentaenoic acid, an ω-3 polyunsaturated fatty acid [“PUFA”], inhigh concentrations.

BACKGROUND OF THE INVENTION

The clinical and pharmaceutical value of eicosapentaenoic acid [“EPA”;cis-5,8,11,14,17-eicosapentaenoic acid; ω-3] are well known (U.S. Pat.Appl. Pub. No. 2009-0093543-A1). Similarly, the advantages of producingEPA in microbes using recombinant means, as opposed to producing EPAfrom natural microbial sources or via isolation from fish oil and marineplankton, are also well recognized.

Although the literature reports a number of recent examples wherebyvarious portions of the ω-3/ω-6 polyunsaturated fatty acid [“PUFA”]biosynthetic pathway, responsible for EPA production, have beenintroduced into plants and non-oleaginous yeast, significant efforts bythe Applicants' Assignee has focused on the use of the oleaginous yeast,Yarrowia lipolytica (U.S. Pat. No. 7,238,482; U.S. Pat. Appl. Pub. No.2006-0115881-A1; U.S. Pat. Appl. Pub. No. 2009-0093543-A1). Oleaginousyeast are defined as those yeast that are naturally capable of oilsynthesis and accumulation, wherein oil accumulation is at least 25% ofthe cellular dry weight.

More specifically, U.S. Pat. Appl. Pub. No. 2006-0115881-A1 demonstratedproduction of 9% EPA of total fatty acids in a recombinant Yarrowialipolytica strain without co-synthesis of γ-linolenic acid [“GLA”; ω-6],by expression of the following genes: Δ9 elongase, Δ8 desaturase, Δ5desaturase, Δ17 desaturase, Δ12 desaturase and C_(16/18) elongase.

U.S. Pat. Appl. Pub. No. 2009-0093543-A1 describes optimized recombinantYarrowia lipolytica strains for EPA production and demonstratedproduction of up to 55.6% EPA of total fatty acids in a recombinant Y.lipolytica strain by expression of the following genes: Δ9 elongase, Δ8desaturase, Δ5 desaturase, Δ17 desaturase, Δ12 desaturase, C_(16/18)elongase and diacylglycerol cholinephosphotransferase, within a hostcell comprising a disruption in the native peroxisome biogenesis factor10 protein (PEX10).

Despite the disclosures cited above, strain improvements are necessaryfor commercial production of EPA that will permit production of high EPAas a weight percent of the total fatty acids in addition to high totallipid content, while minimizing production of intermediate fatty acids,such as linoleic acid [“LA”; ω-6], and byproduct fatty acids in thefinal oil product. Applicants have solved the stated problem byengineering improved optimized strains of Yarrowia lipolytica, whereinthe improvement enables at least one of the following: production of61.8% EPA in the total oil fraction, production of 39.6% total fattyacids as a percent of the dry cell weight, or production of lipidshaving an EPA to LA ratio of 6.1.

SUMMARY OF THE INVENTION

In a first embodiment, the invention concerns an extracted oilcomprising:

(a) at least 50 weight percent of eicosapentaenoic acid measured as aweight percent of total fatty acids; and,

(b) having a ratio of at least 3.1 of eicosapentaenoic acid, measured asa weight percent of total fatty acids, to linoleic acid, measured as aweight percent of total fatty acids.

Preferably, the oil is microbial.

In a second embodiment, the invention concerns an extracted oil of theinvention wherein said oil is extracted from fermented recombinantYarrowia sp. cells engineered for production of eicosapentaenoic acid,wherein said cells comprise:

a) at least at least one multizyme which comprises a polypeptide havingat least one Δ9 elongase linked to at least one Δ8 desaturase;

(b) at least one peroxisome biogenesis factor protein whose expressionhas been down-regulated; and,

(c) at least one recombinant construct comprising a nucleotide sequenceencoding an enzyme selected from the group consisting of malonyl CoAsynthetase and acyl-CoA lysophospholipid acyltransferase.

Preferably, the malonyl CoA synthetase consists essentially of asequence selected from the group consisting of SEQ ID NO:40 and SEQ IDNO:42.

Preferably, the acyl-CoA lysophospholipid acyltransferase consistsessentially of a sequence selected from the group consisting of SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:31 and SEQ IDNO:32.

Preferably, the multizyme linker is selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 and SEQ ID NO:7.

Preferably, the multizyme consists essentially of a sequence selectedfrom the group consisting of: SEQ ID NO:9, SEQ ID NO:11 and SEQ IDNO:13.

In a third embodiment, the invention concerns a blended oil comprisingthe oil of any of claims 1-6 or a derivative thereof.

The blended oil of the invention can also comprise an additionalquantity of a fatty acid selected from the group consisting of: linoleicacid, γ-linolenic, eicosadienoic acid, dihomo-γ-linolenic acid,arachidonic acid, docosatetraenoic acid, ω-6 docosapentaenoic acid,α-linolenic acid, stearidonic acid, eicosatrienoic acid,eicosatetraenoic acid, ω-3 docosapentaenoic acid and docosahexaenoicacid.

In a fourth embodiment, the invention concerns food or feed comprisingthe oil of the invention or a derivative thereof or a blend of the oilor derivative thereof.

In a fifth embodiment, the invention concerns food of the inventionwherein said food is selected from the group consisting of a foodanalog, a functional food, a medical food and a medical nutritional.

In a sixth embodiment, the invention concerns a product comprising theoil of the invention or a derivative thereof or a blend of the oil orderivative thereof, wherein said product is selected from the groupconsisting of a pharmaceutical product, infant formula, dietarysupplement, and animal feed.

In a seventh embodiment, the invention concerns a microbial biomasscomprising the oil of the invention.

In an eighth embodiment, the invention concerns animal feed comprisingthe microbial biomass of the invention.

In a ninth embodiment, the invention concerns animal feed of theinvention wherein the feed is an aquaculture feed.

In a tenth embodiment, the invention concerns oil of the inventionwherein said oil is extracted from a recombinant Yarrowia sp. host cellusing a process selected from the group consisting of: extraction withorganic solvents, sonication and supercritical fluid extraction.

Biological Deposits

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, accession numbersand dates of deposit.

Biological Material Accession No. Date of Deposit Yarrowia lipolyticaY8406 ATCC PTA-10025 May 14, 2009 Yarrowia lipolytica Y8412 ATCCPTA-10026 May 14, 2009 Yarrowia lipolytica Y8259 ATCC PTA-10027 May 14,2009

The biological materials listed above were deposited under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. The listed depositwill be maintained in the indicated international depository for atleast 30 years and will be made available to the public upon the grantof a patent disclosing it. The availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by government action.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1A and FIG. 1B illustrate the ω-3/ω-6 fatty acid biosyntheticpathway, and should be viewed together when considering the descriptionof this pathway below.

FIG. 2 diagrams the development of Yarrowia lipolytica strains Y9481,Y9497 and Y9502, producing greater than 60.9% EPA in the total lipidfraction.

FIG. 3 provides a plasmid map for pY116.

FIG. 4 provides plasmid maps for the following: (A) pZKSL-555A5; and,(B) pZP3-Pa777U.

FIG. 5 provides plasmid maps for the following: (A) pZKUM; and, (B)pZKL2-5mB89C.

FIG. 6 provides plasmid maps for the following: (A) pZKL1-2SR9G85; and,(B) pZSCP-Ma83.

FIG. 7 provides plasmid maps for the following: (A) pZKL4-398F2; and,(B) pZP2-85 m98F.

FIG. 8 provides plasmid maps for the following: (A) pZK16-ML8N; and, (B)pZK16-ML.

FIG. 9 diagrams the development of Yarrowia lipolytica strain Y8672,producing greater than 61.8% EPA in the total lipid fraction.

FIG. 10 provides plasmid maps for the following: (A) pZKL2-5m89C; and,(B) pY201, comprising a chimeric YAT1::ScAle1S::Lip1 gene.

FIG. 11 provides plasmid maps for the following: (A) pY168, comprising achimeric YAT1::YIAle1::Lip1 gene; and, (B) pY208, comprising a chimericYAT1::MaLPAAT1S::Lip1 gene.

FIG. 12 provides plasmid maps for the following: (A) pY207, comprising achimeric YAT1::YILPAAT1::Lip1 gene; and, (B) pY175, comprising achimeric YAT1::CeLPCATS::Lip1 gene.

FIG. 13 provides a comparison of EPA % TFAs, LA % TFAs and the ratio ofEPA % TFAs to LA % TFAs in each of the strains described in theExamples.

FIG. 14 provides plasmid maps for the following: (A) pY222, comprising achimeric YAT1::ScLPAATS::Lip1 gene; and, (B) pY177, comprising achimeric YAT1::YILPAAT1::Lip1 gene.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R.§1.822.

SEQ ID NOs:1-156 are ORFs encoding promoters, genes or proteins (orfragments thereof) or plasmids, as identified in Table 1.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers Nucleic acid ProteinDescription SEQ ID NO. SEQ ID NO. Multizyme linker — 1GAGPARPAGLPPATYYDSLAVMGS Multizyme linker GPARPAGLPPATYYDSLAV — 2Multizyme linker PARPAGLPPATYYDSLAV — 3 Multizyme linkerPTRPAGPPPATYYDSLAV — 4 Multizyme linker — 5PGGPGKPSEIASLPPPIRPVGNPPAAYYDALAT Multizyme linkerPARPAGLPPATYYDSLAVSGRT — 6 Multizyme linker — 7PGGPGKPSEIASLPPPIRPVGNPPAAYYDALATGRT DGLA synthase, comprisingEgD9eS/EgD8M gene 8 9 fusion (2112 bp) (703 AA) DGLA synthase,comprising EaD9eS/EaD8S gene 10 11 fusion (2109 bp) (702 AA) DGLAsynthase, comprising E389D9eS/EgD8M gene 12 13 fusion (2127 bp) (708 AA)Saccharomyces cerevisiae Ale1 (“ScAle1”; also ORF 14 15 “YOR175C”) (1860bp) (619 AA) Yarrowia lipolytica Ale1 (“YlAle1”) 16 17 (1539 bp) (512AA) membrane bound O-acyltransferase motif — 18 M(V/I)LxxKL membranebound O-acyltransferase motif — 19 RxKYYxxW membrane boundO-acyltransferase motif SAxWHG — 20 U.S. Pat. Appl. Pub. No.2008-0145867-A1 motif — 21 EX₁₁WNX₂-[T/V]-X₂W Synthetic Ale1 derivedfrom Saccharomyces 22 23 cerevisiae, codon-optimized for expression in(1870 bp) (619 AA) Yarrowia lipolytica (“ScAle1S”) Caenorhabditiselegans LPCAT (“CeLPCAT”) 24 25 (849 bp) (282 AA) Synthetic LPCATderived from Caenorhabditis 26 27 elegans, codon-optimized forexpression in Yarrowia (859 bp) (282 AA) lipolytica (“CeLPCATS”)Mortierella alpina LPAAT1 (“MaLPAAT1”) 28 29 (945 bp) (314 AA) Yarrowialipolytica LPAAT1 (“YILPAAT1”) 30 31 (1549 bp) (282 AA) Saccharomycescerevisiae LPAAT (“ScLPAAT”; also — 32 ORF “YDL052C”) (303 AA)1-acyl-sn-glycerol-3-phosphate acyltransferase motif — 33 NHxxxxD1-acyl-sn-glycerol-3-phosphate acyltransferase motif — 34 EGTR SyntheticLPAAT1 derived from Mortierella alpina, 35 36 codon-optimized forexpression in Yarrowia lipolytica (955 bp) (314 AA) (“MaLPAAT1S”)Yarrowia lipolytica diacylglycerol 37 38 cholinephosphotransferase gene(“YICPT1”) (1185 bp) (394 AA) Rhizobium leguminosarum bv. viciae 3841malonyl- 39 40 CoA synthetase (GenBank Accession No. (1515 bp) (504 AA)YP_766603) (“rMCS”) Synthetic malonyl-CoA synthetase derived from 41 42Rhizobium leguminosarum bv. viciae 3841 (GenBank (1518 bp) (505 AA)Accession No. YP_766603), codon-optimized for expression in Yarrowialipolytica (“MCS”) Euglena gracilis Δ9 elongase (“EgD9e”) 43 44 (777 bp)(258 AA) Synthetic Δ9 elongase derived from Euglena gracilis, 45 46codon-optimized for expression in Yarrowia lipolytica (777 bp) (258 AA)(“EgD9eS”) Eutreptiella sp. CCMP389 Δ9 elongase (“E389D9e”) 47 48 (792bp) (263 AA) Synthetic Δ9 elongase derived from Eutreptiella sp. 49 50CCMP389 codon-optimized for expression in (792 bp) (263 AA) Yarrowialipolytica (“E389D9eS”) Euglena anabaena UTEX 373 Δ9 elongase 51 52(“EaD9Elo1”) (774 bp) (258 AA) Synthetic Δ9 elongase derived fromEuglena 53 54 anabaena UTEX 373, codon-optimized for (774 bp) (258 AA)expression in Yarrowia lipolytica (“EaD9eS”) Euglena gracilis 48desaturase (“Eg5”or “EgD8”) 55 56 (1271 bp) (421 AA) Synthetic Δ8desaturase derived from Euglena 57 58 gracilis, codon-optimized forexpression in Yarrowia (1272 bp) (422 AA) lipolytica (“D8SF”or “EgD8S”)Synthetic mutant Δ8 desaturase (“EgD8M”), derived 59 60 from Euglenagracilis (“EgD8S”) (1272 bp) (422 AA) Euglena anabaena UTEX 373 Δ8desaturase 61 62 (“EaD8es3”) (1260 bp) (420 AA) Synthetic Δ8 desaturasederived from Euglena 63 64 anabaena UTEX 373, codon-optimized for (1260bp) (420 AA) expression in Yarrowia lipolytica (“EaD8S”) Euglenagracilis Δ5 desaturase (“EgD5”) 65 66 (1350 bp) (449 AA) Synthetic Δ5desaturase derived from Euglena 67 68 gracilis, codon-optimized forexpression in Yarrowia (1350 bp) (449 AA) lipolytica (“EgD5S”) Mutant Δ5desaturase (“EgD5M”), derived from 69 70 Euglena gracilis (“EgD5”) (U.S.Pat. Pub. No. 2010- (1350 bp) (449 AA) 0075386-A1) Synthetic mutant Δ5desaturase (“EgD5SM”), 71 72 derived from Euglena gracilis (“EgD5S”)(U.S. Pat. (1350 bp) (449 AA) Pub. No. 2010-0075386-A1) Peridinium sp.CCMP626 Δ5 desaturase (“RD5”) 73 74 (1392 bp) (463 AA) Synthetic Δ5desaturase derived from Peridinium sp. 75 76 CCMP626, codon-optimizedfor expression in (1392 bp) (463 AA) Yarrowia lipolytica (“RD5S”)Euglena anabaena UTEX 373 Δ5 desaturase 77 78 (“EaD5”) (1362 bp) (454AA) Synthetic Δ5 desaturase derived from Euglena 79 80 anabaena UTEX373, codon-optimized for (1362 bp) (454 AA) expression in Yarrowialipolytica (“EaD5S”) Synthetic mutant Δ5 desaturase (“EaD5SM”), derived81 82 from Euglena anabaena (“EaD5S”) (U.S. Pat. Pub. (1365 bp) (454 AA)No. 2010-0075386-A1) Phytophthora ramorum Δ17 desaturase (“PrD17”) 83 84(1086 bp) (361 AA) Synthetic Δ17 desaturase derived from Phytophthora 8586 ramorum, codon-optimized for expression in (1086 bp) (361 AA)Yarrowia lipolytica (“PrD17S”) Pythium aphanidermatum Δ17 desaturase(“PaD17”) 87 88 (1080 bp) (359 AA) Synthetic Δ17 desaturase derived fromPythium 89 90 aphanidermatum, codon-optimized for expression in (1080bp) (359 AA) Yarrowia lipolytica (“PaD17S”) Fusarium moniliforme Δ12desaturase (“FmD12”) 91 92 (1434 bp) (477 AA) Synthetic Δ12 desaturasederived from Fusarium 93 94 moniliforme, codon-optimized for expressionin (1434 bp) (477 AA) Yarrowia lipolytica (“FmD12S”) Mortierella alpinaC_(16/18) elongase 95 96 (828 bp) (275 AA) Synthetic C_(16/18) elongasederived from Mortierella 97 98 alpina ELO3, codon-optimized forexpression in (828 bp) (275 AA) Yarrowia lipolytica (“ME3S”) Shindou etal. membrane bound O-acyltransferase — 99 motif WHGxxxGYxxxF Shindou etal. membrane bound O-acyltransferase — 100 motif YxxxxF Shindou et al.membrane bound O-acyltransferase — 101 motif YxxxYFxxH U.S. Pat. Appl.Pub. No. 2008-0145867-A1 motif — 102 M[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDGU.S. Pat. Appl. Pub. No. 2008-0145867-A1 motif — 103RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG U.S. Pat. Appl. Pub. No.2008-0145867-A1 motif — 104 SAxWHGxxPGYxx-[T/F]-F Lewin, T. W. et al. &Yamashita et al. 1-acyl-sn- — 105 glycerol-3-phosphate acyltransferasemotif GxxFI-[D/R]-R Lewin, T. W. et al. 1-acyl-sn-glycerol-3-phosphate —106 acyltransferase motif [V/I]-[P/X]-[I/V/L]-[I/V]-P-[V/I] Yamashita etal. 1-acyl-sn-glycerol-3-phosphate — 107 acyltransferase motif IVPIVMYarrowia lipolytica Pex1p (GenBank Accession No. — 108 CAG82178) (1024AA) Yarrowia lipolytica Pex2p — 109 (GenBank Accession No. CAG77647)(381 AA) Yarrowia lipolytica Pex3p (GenBank Accession No. — 110CAG78565) (431 AA) Yarrowia lipolytica Pex3Bp (GenBank Accession No. —111 CAG83356) (395 AA) Yarrowia lipolytica Pex4p (GenBank Accession No.— 112 CAG79130) (153 AA) Yarrowia lipolytica Pex5p (GenBank AccessionNo. — 113 CAG78803) (598 AA) Yarrowia lipolytica Pex6p (GenBankAccession No. — 114 CAG82306) (1024 AA) Yarrowia lipolytica Pex7p(GenBank Accession No. — 115 CAG78389) (356 AA) Yarrowia lipolyticaPex8p (GenBank Accession No. — 116 CAG80447) (671 AA) Yarrowialipolytica Pex10p (GenBank Accession No. — 117 CAG81606) (377 AA)Yarrowia lipolytica Pex12p (GenBank Accession No. — 118 CAG81532) (408AA) Yarrowia lipolytica Pex13p (GenBank Accession No. — 119 CAG81789)(412 AA) Yarrowia lipolytica Pex14p (GenBank Accession No. — 120CAG79323) (380 AA) Yarrowia lipolytica Pex16p (GenBank Accession No. —121 CAG79622) (391 AA) Yarrowia lipolytica Pex17p (GenBank Accession No.— 122 CAG84025) (225 AA) Yarrowia lipolytica Pex19p (GenBank AccessionNo. — 123 AAK84827) (324 AA) Yarrowia lipolytica Pex20p (GenBankAccession No. — 124 CAG79226) (417 AA) Yarrowia lipolytica Pex22p(GenBank Accession No. — 125 CAG77876) (195 AA) Yarrowia lipolyticaPex26p (GenBank Accession No. — 126 NC_006072, antisense translation ofnucleotides (386 AA) 117230-118387) Plasmid pY116 127 — (8739 bp)Plasmid pZKSL-5S5A5 128 — (13975 bp) Plasmid pZP3-Pa777U 129 — (13066bp) Plasmid pZKUM 130 — (4313 bp) Plasmid pZKL2-5mB89C 131 — (15991 bp)Plasmid pZKL1-2SR9G85 132 — (14554 bp) Plasmid pZSCP-Ma83 133 — (15119bp) Plasmid pZKL4-398F2 134 — (14623 bp) Plasmid pZP2-85m98F 135 —(14619 bp) Plasmid pZK16-ML8N 136 — (15262 bp) Plasmid pZK16-ML 137 —(13075bp) Plasmid pZKL2-5m89C 138 — (15799 bp) Plasmid pY201 139 — (9641bp) Escherichia coli LoxP recombination site, recognized 140 — by a Crerecombinase enzyme (34 bp) Primer 798 141 — Primer 799 142 — Primer 800143 — Primer 801 144 — Plasmid pY168 145 — (9320 bp) Plasmid pY208 146 —(8726 bp) Primer 856 147 — Primer 857 148 — Plasmid pY207 149 — (8630bp) Plasmid pY175 150 — (8630 bp) Synthetic LPAAT derived fromSaccharomyces 151 152 cerevisiae, codon-optimized for expression in (926bp) (303 AA) Yarrowia lipolytica (“ScLPAATS”) Plasmid pY222 153 — (7891bp) Primer 869 154 — Primer 870 155 — Plasmid pY177 156 — (9598 bp)

DETAILED DESCRIPTION OF THE INVENTION

Described herein are production host strains of Yarrowia lipolytica thatare capable of producing greater than 50% eicosapentaenoic acid [“EPA”;20:5 ω-3]. Accumulation of this particular polyunsaturated fatty acid[“PUFA”] is accomplished by introduction of a functional ω-3/ω-6 fattyacid biosynthetic pathway comprising proteins with Δ9 elongase, Δ8desaturase, Δ5 desaturase, Δ17 desaturase, Δ12 desaturase and C_(16/18)elongase activities, which thereby enables production of an EPA oil withminimal γ-linolenic acid [“GLA”]. Thus, this disclosure demonstratesthat Y. lipolytica can be engineered to enable commercial production ofEPA and derivatives thereof. Methods of production, and oils therefrom,are also claimed.

PUFAs, such as EPA (or derivatives thereof), are used as dietarysubstitutes, or supplements, particularly infant formulas, for patientsundergoing intravenous feeding or for preventing or treatingmalnutrition. Alternatively, the purified PUFAs (or derivatives thereof)may be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount fordietary supplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use, either human orveterinary.

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with EPA can result notonly in increased levels of EPA, but also downstream products of EPAsuch as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes),docosapentaenoic acid [“DPA”; cis-7,10,13,16,19-docosapentaenoic; 22:5ω-3] and docosahexaenoic acid [“DHA”;cis-4,7,10,13,16,19-docosahexaenoic acid; 22:6 ω-3]. Complex regulatorymechanisms can make it desirable to combine various PUFAs, or adddifferent conjugates of PUFAs, in order to prevent, control or overcomesuch mechanisms to achieve the desired levels of specific PUFAs in anindividual.

Alternately, PUFAs, or derivatives thereof, made by the methodologydisclosed herein can be utilized in the synthesis of animal andaquaculture feeds, such as dry feeds, semi-moist and wet feeds, sincethese formulations generally require at least 1-2% of the nutrientcomposition to be ω-3 and/or ω-6 PUFAs.

All patents, patent applications, and publications cited herein areincorporated by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated as “ORF”.

“Polymerase chain reaction” is abbreviated as “PCR”.

“American Type Culture Collection” is abbreviated as “ATCC”.

“Polyunsaturated fatty acid(s)” is abbreviated as “PUFA(s)”.

“Triacylglycerols” are abbreviated as “TAGs”.

“Co-enzyme A” is abbreviated as “CoA”.

“Total fatty acids” are abbreviated as “TFAs”.

“Fatty acid methyl esters” are abbreviated as “FAMEs”.

“Dry cell weight” is abbreviated as “DCW”.

As used herein the term “invention” or “present invention” is intendedto refer to all aspects and embodiments of the invention as described inthe claims and specification herein and should not be read so as to belimited to any particular embodiment or aspect.

The term “food product” refers to any food generally suitable for humanconsumption. Typical food products include, but are not limited to: meatproducts, cereal products, baked foods, snack foods, dairy products,beverages and the like. The terms “food analog”, “functional food”,“medical food” and “medical nutritional” are defined as in U.S. Pat.Appl. Pub. No. 2006-0115881-A1.

The term “pharmaceutical” as used herein means a compound or substancewhich if sold in the United States would be controlled by Section 503 or505 of the Federal Food, Drug and Cosmetic Act.

The term “infant formula” means a food which is designed exclusively forconsumption by the human infant by reason of its simulation of humanbreast milk. Typical commercial examples of infant formula include, butare not limited to: Similac® and Isomil®.

The term “dietary supplement” refers to a product that: (i) is intendedto supplement the diet and thus is not represented for use as aconventional food or as a sole item of a meal or the diet; (ii) containsone or more dietary ingredients (including, e.g., vitamins, minerals,herbs or other botanicals, amino acids, enzymes and glandulars) or theirconstituents; (iii) is intended to be taken by mouth as a pill, capsule,tablet, or liquid; and, (iv) is labeled as being a dietary supplement.

The term “animal feed” refers to feeds intended exclusively forconsumption by animals, including domestic animals such as pets, farmanimals, etc. or for animals raised for the production of food, such asfor e.g., fish farming. The terms “aquaculture feed”, “aquafeed” and“feed nutrient” are as defined in U.S. Pat. Appl. Pub. No.2006-0115881-A1.

As used herein the term “biomass” refers specifically to spent or usedyeast cellular material from the fermentation of a recombinantproduction host producing EPA in commercially significant amounts,wherein the preferred production host is a recombinant strain of theoleaginous yeast, Yarrowia lipolytica. The biomass may be in the form ofwhole cells, whole cell lysates, homogenized cells, partially hydrolyzedcellular material, and/or partially purified cellular material (e.g.,microbially produced oil).

The term “lipids” refer to any fat-soluble (i.e., lipophilic),naturally-occurring molecule. A general overview of lipids is providedin U.S. Pat. Appl. Pub. No. 2009-0093543-A1 (see Table 2 therein).

The term “glycerophospholipids” refers to a broad class of molecules,having a glycerol core with fatty acids at the sn-1 position and sn-2position, and a polar head group (e.g., phosphate, choline,ethanolamine, glycerol, inositol, serine, cardiolipin) joined at thesn-3 position via a phosphodiester bond. Glycerophospholipids thusinclude phosphatidic acid [“PA”], phosphatidylcholines [“PC”],phosphatidylethanolamines [“PE”], phosphatidylglycerols [“PG”],phosphatidylinositols [“PI”], phosphatidylserines [“PS”] andcardiolipins [“CL”]. Glycerophospholipids possess tremendous diversity,not only resulting from variable phosphoyl head groups, but also as aresult of differing chain lengths and degrees of saturation of theirfatty acids. Generally, saturated and monounsaturated fatty acids areesterified at the sn-1 position, while polyunsaturated fatty acids areesterified at the sn-2 position.

“Lysophospholipids” are derived from glycerophospholipids, bydeacylation of the sn-2 position fatty acid. Lysophospholipids include,e.g., lysophosphatidic acid [“LPA”], lysophosphatidylcholine [“LPC”],lysophosphatidyletanolamine [“LPE”], lysophosphatidylserine [“LPS”],lysophosphatidylglycerol [“LPG”] and lysophosphatidylinositol [“LPI”].

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. In oleaginous organisms, oil constitutes amajor part of the total lipid. “Oil” is composed primarily oftriacylglycerols [“TAGs”] but may also contain other neutral lipids,phospholipids and free fatty acids. The fatty acid composition in theoil and the fatty acid composition of the total lipid are generallysimilar; thus, an increase or decrease in the concentration of PUFAs inthe total lipid will correspond with an increase or decrease in theconcentration of PUFAs in the oil, and vice versa.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and are so called because at cellular pH, thelipids bear no charged groups. Generally, they are completely non-polarwith no affinity for water. Neutral lipids generally refer to mono-,di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or triacylglycerol, respectively, orcollectively, acylglycerols. A hydrolysis reaction must occur to releasefree fatty acids from acylglycerols.

The term “triacylglycerols” [“TAGs”] refers to neutral lipids composedof three fatty acyl residues esterified to a glycerol molecule. TAGs cancontain long chain PUFAs and saturated fatty acids, as well as shorterchain saturated and unsaturated fatty acids.

The term “total fatty acids” [“TFAs”] herein refer to the sum of allcellular fatty acids that can be derivitized to fatty acid methyl esters[“FAMEs”] by the base transesterification method (as known in the art)in a given sample, which may be the biomass or oil, for example. Thus,total fatty acids include fatty acids from neutral lipid fractions(including diacylglycerols, monoacylglycerols and TAGs) and from polarlipid fractions (including, e.g., the PC and the PE fractions) but notfree fatty acids.

The term “total lipid content” of cells is a measure of TFAs as apercent of the dry cell weight [“DCW”], although total lipid content canbe approximated as a measure of FAMEs as a percent of the DCW [“FAMEs %DCW”]. Thus, total lipid content [“TFAs % DCW”] is equivalent to, e.g.,milligrams of total fatty acids per 100 milligrams of DCW.

The concentration of a fatty acid in the total lipid is expressed hereinas a weight percent of TFAs [“TFAs”], e.g., milligrams of the givenfatty acid per 100 milligrams of TFAs. Unless otherwise specificallystated in the disclosure herein, reference to the percent of a givenfatty acid with respect to total lipids is equivalent to concentrationof the fatty acid as % TFAs (e.g., % EPA of total lipids is equivalentto EPA % TFAs).

In some cases, it is useful to express the content of a given fattyacid(s) in a cell as its weight percent of the dry cell weight [“%DCW”]. Thus, for example, EPA % DCW would be determined according to thefollowing formula: (EPA % TFAs)*(TFAs % DCW)]/100. The content of agiven fatty acid(s) in a cell as its weight percent of the dry cellweight [“% DCW”] can be approximated, however, as: (EPA % TFAs)*(FAMEsDCW)]/100.

The terms “lipid profile” and “lipid composition” are interchangeableand refer to the amount of individual fatty acids contained in aparticular lipid fraction, such as in the total lipid or the oil,wherein the amount is expressed as a weight percent of TFAs. The sum ofeach individual fatty acid present in the mixture should be 100.

The term “extracted oil” refers to an oil that has been separated fromother cellular materials, such as the microorganism in which the oil wassynthesized. Extracted oils are obtained through a wide variety ofmethods, the simplest of which involves physical means alone. Forexample, mechanical crushing using various press configurations (e.g.,screw, expeller, piston, bead beaters, etc.) can separate oil fromcellular materials. Alternately, oil extraction can occur via treatmentwith various organic solvents (e.g., hexane), via enzymatic extraction,via osmotic shock, via ultrasonic extraction, via supercritical fluidextraction (e.g., CO₂ extraction), via saponification and viacombinations of these methods. An extracted oil does not require that itis not necessarily purified or further concentrated. The extracted oilsdescribed herein will comprise at least 50 EPA % TFAs.

The term “blended oil” refers to an oil that is obtained by admixing, orblending, the extracted oil described herein with any combination of, orindividual, oil to obtain a desired composition. Thus, for example,types of oils from different microbes can be mixed together to obtain adesired PUFA composition. Alternatively, or additionally, thePUFA-containing oils disclosed herein can be blended with fish oil,vegetable oil or a mixture of both to obtain a desired composition.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂, although bothlonger and shorter chain-length acids are known. The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon [“C”] atoms in the particular fatty acid and Y is thenumber of double bonds. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” [“PUFAs”], and “omega-6 fatty acids” [“ω-6” or “n-6”]versus “omega-3 fatty acids” [“ω-3” or “n-3”] are provided in U.S. Pat.No. 7,238,482, which is hereby incorporated herein by reference.

Nomenclature used to describe PUFAs herein is given in Table 2. In thecolumn titled “Shorthand Notation”, the omega-reference system is usedto indicate the number of carbons, the number of double bonds and theposition of the double bond closest to the omega carbon, counting fromthe omega carbon, which is numbered 1 for this purpose. The remainder ofthe Table summarizes the common names of ω-3 and ω-6 fatty acids andtheir precursors, the abbreviations that will be used throughout thespecification and the chemical name of each compound.

TABLE 2 Nomenclature of Polyunsaturated Fatty Acids And PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9, 12-octadecadienoic 18:2 ω-6γ-Linolenic GLA cis-6, 9, 12-octadecatrienoic 18:3 ω-6 Eicosadienoic EDAcis-11, 14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLA cis-8, 11,14-eicosatrienoic 20:3 ω-6 Linolenic Arachidonic ARA cis-5, 8, 11, 14-20:4 ω-6 eicosatetraenoic α-Linolenic ALA cis-9, 12, 15- 18:3 ω-3octadecatrienoic Stearidonic STA cis-6, 9, 12, 15- 18:4 ω-3octadecatetraenoic Eicosatrienoic ETrA cis-11, 14, 17-eicosatrienoic20:3 ω-3 Sciadonic SCI cis-5, 11, 14-eicosatrienoic 20:3b ω-6Juniperonic JUP cis-5, 11, 14, 17- 20:4b ω-3 eicosatetraenoic Eicosa-ETA cis-8, 11, 14, 17- 20:4 ω-3 tetraenoic eicosatetraenoic Eicosa- EPAcis-5, 8, 11, 14, 17- 20:5 ω-3 pentaenoic eicosapentaenoic Docosa- DTAcis-7, 10, 13, 16- 22:4 ω-6 tetraenoic docosatetraenoic Docosa- DPAn-6cis-4, 7, 10, 13, 16- 22:5 ω-6 pentaenoic docosapentaenoic Docosa- DPAcis-7, 10, 13, 16, 19- 22:5 ω-3 pentaenoic docosapentaenoic Docosa- DHAcis-4, 7, 10, 13, 16, 19- 22:6 ω-3 hexaenoic docosahexaenoic

The term “PUFA biosynthetic pathway” refers to a metabolic process thatconverts oleic acid to ω-6 fatty acids such as LA, EDA, GLA, DGLA, ARA,DTA and DPAn-6 and ω-3 fatty acids such as ALA, STA, ETrA, ETA, EPA, DPAand DHA. This process is well described in the literature (e.g., seeU.S. Pat. Appl. Pub. No. 2006-0115881-A1). Briefly, this processinvolves elongation of the carbon chain through the addition of carbonatoms and desaturation of the molecule through the addition of doublebonds, via a series of special elongation and desaturation enzymestermed “PUFA biosynthetic pathway enzymes” that are present in theendoplasmic reticulum membrane. More specifically, “PUFA biosyntheticpathway enzymes” refer to any of the following enzymes (and genes whichencode said enzymes) associated with the biosynthesis of a PUFA,including: Δ4 desaturase, Δ5 desaturase, Δ6 desaturase, Δ12 desaturase,Δ15 desaturase, Δ17 desaturase, Δ9 desaturase, Δ8 desaturase, Δ9elongase, C_(14/16) elongase, C_(16/18) elongase, C_(18/20) elongaseand/or C_(20/22) elongase.

The term “Δ9 elongase/Δ8 desaturase pathway” will refer to a PUFAbiosynthetic pathway that minimally includes at least one Δ9 elongaseand at least one Δ8 desaturase, thereby enabling biosynthesis of DGLAand/or ETA from LA and ALA, respectively, with EDA and/or ETrA asintermediate fatty acids. With expression of other desaturases andelongases, ARA, DTA, DPAn-6, EPA, DPA and DHA may also be synthesized.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are: Δ8 desaturases, Δ5 desaturases, Δ17desaturases and Δ12 desaturases. Other useful desaturases can include Δ4desaturases, Δ6 desaturases, Δ15 desaturases and Δ9 desaturases.

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid 2 carbons longer than the fattyacid substrate that the elongase acts upon. This process of elongationoccurs in a multi-step mechanism in association with fatty acidsynthase, as described in Intl. App. Pub. No. WO 2005/047480. Examplesof reactions catalyzed by elongase systems are the conversion of GLA toDGLA, STA to ETA, ARA to DTA and EPA to DPA. In general, the substrateselectivity of elongases is somewhat broad but segregated by both chainlength and the degree and type of unsaturation. For example, a C_(14/16)elongase will utilize a C₁₄ substrate (e.g., myristic acid), a C_(16/18)elongase will utilize a C₁₆ substrate (e.g., palmitate), a C_(18/20)elongase will utilize a C₁₈ substrate (e.g., GLA, STA) and a C_(20/22)elongase [also referred to as a Δ5 elongase or C20 elongase] willutilize a C₂₀ substrate (e.g., ARA, EPA). For the purposes herein, twodistinct types of C_(18/20) elongases can be defined: a Δ6 elongase willcatalyze conversion of GLA and STA to DGLA and ETA, respectively, whilea Δ9 elongase is able to catalyze the conversion of LA and ALA to EDAand ETrA, respectively.

The term “multizyme” or “fusion protein” refers to a single polypeptidehaving at least two independent and separable enzymatic activities,wherein the first enzymatic activity is preferably linked to the secondenzymatic activity (U.S. Pat. Appl. Pub. No. 2008-0254191-A1). The“link” or “bond” between the at least two independent and separableenzymatic activities is minimally comprised of a single polypeptidebond, although the link may also be comprised of one amino acid residue,such as proline, or a polypeptide comprising at least one proline aminoacid residue. Preferred linkers are selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 and SEQ ID NO:7.

The term “DGLA synthase” refers to a multizyme, wherein a Δ9 elongase islinked to a Δ8 desaturase. The term “EgD9eS/EgD8M” refers to a DGLAsynthase (SEQ ID NOs:8 and 9) created by linking the Δ9 elongase“EgD9eS” (U.S. Pat. No. 7,645,604) to the Δ8 desaturase “EgD8M” (U.S.Pat. No. 7,709,239) with a linker sequence (i.e., SEQ ID NO:1[GAGPARPAGLPPATYYDSLAVMGS]; U.S. Pat. Appl. Pub. No. 2008-0254191-A1).Similarly, the term “EaD9eS/EaD8S” refers to a DGLA synthase (SEQ IDNOs:10 and 11) created by linking the Δ9 elongase “EaD9eS” (U.S. Pat.Appl. Pub. No. 2008-0254522-A1) to the Δ8 desaturase “EaD8S” (U.S. Pat.Appl. Pub. No. 2008-0254521-A1) with the linker sequence set forth asSEQ ID NO:1. And, the term “E389D9eS/EgD8M” refers to a DGLA synthase(SEQ ID NOs:12 and 13) created by linking the Δ9 elongase “E389D9eS”(U.S. Pat. No. 7,645,604) to the Δ8 desaturase “EgD8M” (supra) with thelinker sequence set forth as SEQ ID NO:1.

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme, such as adesaturase, elongase or multizyme, can convert substrate to product. Theconversion efficiency is measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “C₁₈ to C₂₀ elongation conversion efficiency” refers to theefficiency by which C_(18/20) elongases can convert C₁₈ substrates(i.e., LA, ALA, GLA, STA) to C₂₀ products (i.e., EDA, ETrA, DGLA, ETA).These C_(18//20) elongases can be either Δ9 elongases or Δ6 elongases.

The term “Δ9 elongation conversion efficiency” refers to the efficiencyby which Δ9 elongase can convert C₁₈ substrates (i.e., LA, ALA) to C₂₀products (i.e., EDA, ETrA).

The term “acyltransferase” refers to an enzyme responsible fortransferring an acyl group from a donor lipid to an acceptor lipidmolecule.

The term “acyl-CoA:lysophospholipid acyltransferase” [“LPLAT”] refers toa broad class of acyltransferases, having the ability to acylate avariety of lysophospholipid substrates at the sn-2 position. Morespecifically, LPLATs include LPA acyltransferases [“LPAATs”] having theability to catalyze conversion of LPA to PA, LPC acyltransferases[“LPCATs”] having the ability to catalyze conversion of LPC to PC, LPEacyltransferases [“LPEATs”] having the ability to catalyze conversion ofLPE to PE, LPS acyltransferases [“LPLATs”] having the ability tocatalyze conversion of LPS to PS, LPG acyltransferases [“LPGATs”] havingthe ability to catalyze conversion of LPG to PG, and LPIacyltransferases [“LPIATs”] having the ability to catalyze conversion ofLPI to PI. Standardization of LPLAT nomenclature has not beenformalized, so various other designations are used in the art (forexample, LPAATs have also been referred to asacyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferases,1-acyl-sn-glycerol-3-phosphate acyltransferases and/or1-acylglycerolphosphate acyltransferases [“AGPATs”] and LPCATs are oftenreferred to as acyl-CoA:1-acyl lysophosphatidyl-cholineacyltransferases). Additionally, it is important to note that someLPLATs, such as the Saccharomyces cerevisiae Ale1 (ORF YOR1750; SEQ IDNO:15), have broad specificity and thus a single enzyme may be capableof catalyzing several LPLAT reactions, including LPAAT, LPCAT and LPEATreactions (Tamaki, H. et al., J. Biol. Chem., 282:34288-34298 (2007);Stahl, U. et al., FEBS Letters, 582:305-309 (2008); Chen, Q. et al.,FEBS Letters, 581:5511-5516 (2007); Benghezal, M. et al., J. Biol.Chem., 282:30845-30855 (2007); Riekhof, et al., J. Biol. Chem.,282:28344-28352 (2007)).

More specifically, the term “polypeptide having at leastlysophosphtidylcholine acyltransferase [“LPCAT”] activity” will refer tothose enzymes capable of catalyzing the reaction:acyl-CoA+1-acyl-sn-glycero-3-phosphocholine→CoA+1,2-diacyl-sn-glycero-3-phosphocholine(EC 2.3.1.23). LPCAT activity has been described in two structurallydistinct protein families, i.e., the LPAAT protein family (Hishikawa, etal., Proc. Natl. Acad. Sci. U.S.A., 105:2830-2835 (2008); Intl. App.Pub. No. WO 2004/076617) and the ALE1 protein family (Tamaki, H. et al.,supra; Ståhl, U. et al., supra; Chen, Q. et al., supra; Benghezal, M. etal., supra; Riekhof, et al., supra).

The term “LPCAT” refers to a protein of the ALE1 protein family that: 1)has LPCAT activity (EC 2.3.1.23) and shares at least about 45% aminoacid identity, based on the Clustal W method of alignment, when comparedto an amino acid sequence selected from the group consisting of SEQ IDNO:15 (ScAle1) and SEQ ID NO:17 (YIAle1); and/or 2) has LPCAT activity(EC 2.3.1.23) and has at least one membrane bound O-acyltransferase[“MBOAT”] protein family motif selected from the group consisting of:M(V/I)LxxKL (SEQ ID NO:18), RxKYYxxW (SEQ ID NO:19), SAxWHG (SEQ IDNO:20) and EX₁₁WNX₂— [T/V]-X₂W (SEQ ID NO:21). Examples of ALE1polypeptides include ScAle1 and YIAIe1.

The term “ScAle1” refers to a LPCAT (SEQ ID NO:15) isolated fromSaccharomyces cerevisiae (ORF “YOR175C”), encoded by the nucleotidesequence set forth as SEQ ID NO:14. In contrast, the term “ScAle1S”refers to a synthetic LPCAT derived from S. cerevisiae that iscodon-optimized for expression in Yarrowia lipolytica (i.e., SEQ IDNOs:22 and 23).

The term “YIAIe1” refers to a LPCAT (SEQ ID NO:17) isolated fromYarrowia lipolytica, encoded by the nucleotide sequence set forth as SEQID NO:16.

The term “LPCAT” also refers to a protein that has LPCAT activity (EC2.3.1.23) and shares at least about 90% amino acid identity, based onthe Clustal W method of alignment, when compared to an amino acidsequence as set forth in SEQ ID NO:25 (CeLPCAT).

The term “CeLPCAT” refers to a LPCAT enzyme (SEQ ID NO:25) isolated fromCaenorhabditis elegans, encoded by the nucleotide sequence set forth asSEQ ID NO:24. In contrast, the term “CeLPCATS” refers to a syntheticLPCAT derived from C. elegans that is codon-optimized for expression inYarrowia lipolytica (i.e., SEQ ID NOs:26 and 27).

The term “polypeptide having at least lysophosphatidic acidacyltransferase [“LPAAT”] activity” will refer to those enzymes capableof catalyzing the reaction: acyl-CoA+1-acyl-sn-glycerol3-phosphate→CoA+1,2-diacyl-sn-glycerol 3-phosphate (EC 2.3.1.51).

The term “LPAAT” refers to a protein that: 1) has LPAAT activity andshares at least about 43.9% amino acid identity, based on the Clustal Wmethod of alignment, when compared to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:29 (MaLPAAT1), SEQ ID NO:31(YILPAAT1) and SEQ ID NO:32 (ScLPAAT1); and/or, 2) has LPAAT activityand has at least one 1-acyl-sn-glycerol-3-phosphate acyltransferasefamily motif selected from the group consisting of: NHxxxxD (SEQ IDNO:33) and EGTR (SEQ ID NO:34). Examples of LPAAT polypeptides includeScLPAAT, MaLPAAT1 and YILPAAT1.

The term “ScLPAAT” refers to a LPAAT (SEQ ID NO:32) isolated fromSaccharomyces cerevisiae (ORF “YDL052C”). The term “MaLPAAT1” refers toa LPAAT (SEQ ID NO:29) isolated from Mortierella alpina, encoded by thenucleotide sequence set forth as SEQ ID NO:28. In contrast, the term“MaLPAAT1S” refers to a synthetic LPAAT derived from M. alpina that iscodon-optimized for expression in Yarrowia lipolytica (i.e., SEQ IDNOs:35 and 36).

The term “YILPAAT1” refers to a LPAAT (SEQ ID NO:31) isolated fromYarrowia lipolytica, encoded by the nucleotide sequence set forth as SEQID NO:30.

The term “ortholog” refers to a homologous protein from a differentspecies that evolved from a common ancestor protein as evidenced bybeing in one Glade of phylogenetic tree analysis and that catalyzes thesame enzymatic reaction.

The term “diacylglycerol cholinephosphotransferase” refers to an enzyme(EC 2.7.8.2) that catalyses the synthesis of phosphatidylcholines fromCDP-choline and 1,2-diacylglycerols. This enzyme is part of theCDP-choline pathway, responsible for phosphatidylcholine [“PC”]biosynthesis.

The term “YICPT1” refers to a diacylglycerol cholinephosphotransferaseenzyme (SEQ ID NO:38) isolated from Yarrowia lipolytica, encoded by SEQID NO:37. YICPT1 is described in Intl. App. Pub. No. WO 2006/052870 (seealso Gen Bank Accession No. XM_(—)501703 (YALI0C10989g)).

The term “malonic acid”, also referred to as propanedioic acid accordingto International Union of Pure and Applied Chemistry [“IUPAC”]systematic nomenclature, refers to a dicarboxylic acid having thechemical structure set forth as CH₂(COOH)₂. The malonate orpropanedioate ion is derived from malonic acid by loss of two hydrogenions (i.e., CH₂(COO)₂ ²⁻). Salts and esters of malonic acid include, butare not limited to, diethyl malonate [(C₂H₅)₂(C₃H₂O₄)], dimethylmalonate [(CH₃)₂(C₃H₂O₄)] and disodium malonate [Na₂(C₃H₂O₄)].

“Malonates” refer to the ionised form of malonic acid, as well as itsesters and salts. All of these are referred to herein collectively as“malonates”.

“Malonyl-CoA” [CAS Registry No. 524-14-1] refers to an acyl thioesterthat can be formed by the carboxylation of acetyl-CoA to malonyl-CoA.Alternatively, malonyl-CoA is produced enzymatically from the substratemalonate, via a malonyl-CoA synthetase.

“Malonyl-CoA synthetase” [EC 6.2.1.-] catalyzes the following enzymaticreaction: malonate+ATP+CoA→malonyl-CoA+AMP+pyrophosphate (PPi). Theenzyme was first purified from malonate-grown Pseudomonas fluorescens(Kim, Y. S, and S. K. Bang, J. Biol. Chem., 260:5098-5104 (1985)),although various Rhizobia homologs have since been isolated frombacteroids within legume nodules (see, for example, Kim, Y. S, and H. Z.Chae, Biochem. J., 273:511-516 (1991) and Kim, Y. S. and S. W. Kang,Biochem. J., 297:327-333 (1994)).

As used herein, the term “rMCS” refers to a gene (SEQ ID NO:39) encodingmalonyl-CoA synthetase (SEQ ID NO:40) isolated from Rhizobiumleguminosarum bv. viciae 3841 (GenBank Accession No. YP_(—)766603).Similarly, the term “MCS” refers to a synthetic gene encodingmalonyl-CoA synthetase derived from Rhizobium leguminosarum bv. viciae3841 that is codon-optimized for expression in Yarrowia lipolytica(i.e., SEQ ID NOs:41 and 42).

The term “peroxisomes” refers to ubiquitous organelles found in alleukaryotic cells. They have a single lipid bilayer membrane thatseparates their contents from the cytosol and that contains variousmembrane proteins essential to the functions described below.Peroxisomes selectively import proteins via an “extended shuttlemechanism”. More specifically, there are at least 32 known peroxisomalproteins, called peroxins, which participate in the process of importingproteins by means of ATP hydrolysis through the peroxisomal membrane.Once cellular proteins are imported into the peroxisome, they aretypically subjected to some means of degradation. For example,peroxisomes contain oxidative enzymes, such as e.g., catalase, D-aminoacid oxidase and uric acid oxidase, that enable degradation ofsubstances that are toxic to the cell. Alternatively, peroxisomesbreakdown fatty acid molecules to produce free molecules of acetyl-CoAwhich are exported back to the cytosol, in a process called β-oxidation.

The terms “peroxisome biogenesis factor protein”, “peroxin” and “Pexprotein” are interchangeable and refer to proteins involved inperoxisome biogenesis and/or that participate in the process ofimporting cellular proteins by means of ATP hydrolysis through theperoxisomal membrane. The acronym of a gene that encodes any of theseproteins is “Pex gene”. A system for nomenclature is described by Distelet al., J. Cell Biol., 135:1-3 (1996). At least 32 different Pex geneshave been identified so far in various eukaryotic organisms. Many Pexgenes have been isolated from the analysis of mutants that demonstratedabnormal peroxisomal functions or structures. Based on a review by Kiel,J. A. K. W., et al. (Traffic, 7:1291-1303 (2006)), wherein in silicoanalysis of the genomic sequences of 17 different fungal species wasperformed, the following Pex proteins were identified: Pex1p, Pex2p,Pex3p, Pex3 Bp, Pex4p, Pex5p, Pex5 Bp, Pex5 Cp, Pex5/20p, Pex6p, Pex7p,Pex8p, Pex10p, Pex12p, Pex13p, Pex14p, Pex15p, Pex16p, Pex17p,Pex14/17p, Pex18p, Pex19p, Pex20p, Pex21p, Pex21 Bp, Pex22p, Pex22p-likeand Pex26p. Collectively, these proteins will be referred to herein as“Pex proteins”, encoded by “Pex genes”.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The term “down-regulated” in or in connection with at least oneperoxisome biogenesis factor protein refers to reduction in, orabolishment of, the activity of a native peroxisome biogenesis factorprotein, as compared to the activity of the wildtype protein.Down-regulation typically occurs when a native Pex gene has a“disruption”, referring to an insertion, deletion, or targeted mutationwithin a portion of that gene, that results in either a complete geneknockout such that the gene is deleted from the genome and no protein istranslated or a translated Pex protein having an insertion, deletion,amino acid substitution or other targeted mutation. The location of thedisruption in the protein may be, for example, within the N-terminalportion of the protein or within the C-terminal portion of the protein.The disrupted Pex protein will have impaired activity with respect tothe Pex protein that was not disrupted, and can be non-functional.Down-regulation that results in low or lack of expression of the Pexprotein, could also result via manipulating the regulatory sequences,transcription and translation factors and/or signal transductionpathways or by use of sense, antisense or RNAi technology, etc.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of oil (Weete, In: Fungal Lipid Biochemistry,2nd Ed., Plenum, 1980). The term “oleaginous yeast” refers to thosemicroorganisms classified as yeasts that can make oil. Generally, thecellular oil content of oleaginous microorganisms follows a sigmoidcurve, wherein the concentration of lipid increases until it reaches amaximum at the late logarithmic or early stationary growth phase andthen gradually decreases during the late stationary and death phases(Yongmanitchai and Ward, Appl. Environ. Microbiol., 57:419-25 (1991)).It is not uncommon for oleaginous microorganisms to accumulate in excessof about 25% of their dry cell weight as oil. Examples of oleaginousyeast include, but are no means limited to, the following genera:Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,Trichosporon and Lipomyces.

The term “fermentable carbon source” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbon sourcesinclude, but are not limited to: monosaccharides, oligosaccharides,polysaccharides, alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleic acid fragment” and “isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410(1993)). In general, a sequence of ten or more contiguous amino acids orthirty or more nucleotides is necessary in order to identify putativelya polypeptide or nucleic acid sequence as homologous to a known proteinor gene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation, such as in situ hybridization ofbacterial colonies or bacteriophage plaques. In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These oligonucleotide building blocks are annealed and thenligated to form gene segments that are then enzymatically assembled toconstruct the entire gene. Accordingly, the genes can be tailored foroptimal gene expression based on optimization of nucleotide sequence toreflect the codon bias of the host cell. The skilled artisan appreciatesthe likelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cell,where sequence information is available. For example, the codon usageprofile for Yarrowia lipolytica is provided in U.S. Pat. No. 7,125,672.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and which may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes inserted into a non-native organism, native genes introduced intoa new location within the native host, or chimeric genes. A “transgene”is a gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, enhancers, silencers, 5′ untranslated leader sequence (e.g.,between the transcription start site and the translation initiationcodon), introns, polyadenylation recognition sequences, RNA processingsites, effector binding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The terms “3′ non-coding sequence” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence. That is, the coding sequence is under thetranscriptional control of the promoter. Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA. Expression mayalso refer to translation of mRNA into a polypeptide.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism. The nucleic acid molecule may be a plasmid thatreplicates autonomously, for example, or, it may integrate into thegenome of the host organism. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” or “recombinant”or “transformed” organisms or “transformant”.

“Stable transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance (i.e.,the nucleic acid fragment is “stably integrated”). In contrast,“transient transformation” refers to the transfer of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes that are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA fragments.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction that is capable of introducing anexpression cassette(s) into a cell.

The term “expression cassette” refers to a fragment of DNA comprisingthe coding sequence of a selected gene and regulatory sequencespreceding (5′ non-coding sequences) and following (3′ non-codingsequences) the coding sequence that are required for expression of theselected gene product. Thus, an expression cassette is typicallycomposed of: 1) a promoter sequence; 2) a coding sequence (i.e., ORF)and, 3) a 3′ untranslated region (i.e., a terminator) that, ineukaryotes, usually contains a polyadenylation site. The expressioncassette(s) is usually included within a vector, to facilitate cloningand transformation. Different expression cassettes can be transformedinto different organisms including bacteria, yeast, plants and mammaliancells, as long as the correct regulatory sequences are used for eachhost.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and, 5) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience, Hoboken, N.J. (1987).

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inU.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chainsaturated and unsaturated fatty acid derivates, which are formed throughthe action of elongases and desaturases (FIG. 1).

A wide spectrum of fatty acids (including saturated and unsaturatedfatty acids and short-chain and long-chain fatty acids) can beincorporated into TAGs, the primary storage unit for fatty acids. In themethods and host cells described herein, incorporation of EPA into TAGsis most desirable, although the structural form of the EPA is notlimiting (thus, for example, the EPA may exist in the total lipids asfree fatty acids or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids).

Although most PUFAs are incorporated into TAGs as neutral lipids and arestored in lipid bodies, it is important to note that a measurement ofthe total PUFAs within an oleaginous organism should minimally includethose PUFAs that are located in the PC, PE and TAG fractions.

The metabolic process wherein oleic acid is converted to EPA involveselongation of the carbon chain through the addition of carbon atoms anddesaturation of the molecule through the addition of double bonds. Thisrequires a series of special desaturation and elongation enzymes presentin the endoplasmic reticulum membrane. However, as seen in FIG. 1 and asdescribed below, multiple alternate pathways exist for EPA production.

Specifically, FIG. 1 depicts the pathways described below. All pathwaysrequire the initial conversion of oleic acid to linoleic acid [“LA”],the first of the ω-6 fatty acids, by a Δ12 desaturase. Then, using the“Δ9 elongase/Δ8 desaturase pathway” and LA as substrate, long-chain ω-6fatty acids are formed as follows: 1) LA is converted to eicosadienoicacid [“EDA”] by a Δ9 elongase; 2) EDA is converted to dihomo-γ-linolenicacid [“DGLA”] by a Δ8 desaturase; 3) DGLA is converted to arachidonicacid [“ARA”] by a Δ5 desaturase; 4) ARA is converted to docosatetraenoicacid [“DTA”] by a C_(20/22) elongase; and, 5) DTA is converted todocosapentaenoic acid [“DPAn-6”] by a Δ4 desaturase.

The “Δ9 elongase/Δ8 desaturase pathway” can also use α-linolenic acid[“ALA”] as substrate to produce long-chain ω-3 fatty acids asfollows: 1) LA is converted to ALA, the first of the ω-3 fatty acids, bya Δ15 desaturase; 2) ALA is converted to eicosatrienoic acid [“ETrA”] bya Δ9 elongase; 3) ETrA is converted to eicosatetraenoic acid [“ETA”] bya Δ8 desaturase; 4) ETA is converted to eicosapentaenoic acid [“EPA”] bya Δ5 desaturase; 5) EPA is converted to docosapentaenoic acid [“DPA”] bya C_(20/22) elongase; and, 6) DPA is converted to docosahexaenoic acid[“DHA”] by a Δ4 desaturase. Optionally, ω-6 fatty acids may be convertedto ω-3 fatty acids. For example, ETA and EPA are produced from DGLA andARA, respectively, by Δ17 desaturase activity. Advantageously for thepurposes herein, the Δ9 elongase/Δ8 desaturase pathway enablesproduction of an EPA oil that lacks significant amounts of γ-linolenicacid [“GLA”].

Alternate pathways for the biosynthesis of ω-3/ω-6 fatty acids utilize aΔ6 desaturase and C_(18/20) elongase, that is, the “Δ6 desaturase/Δ6elongase pathway”. More specifically, LA and ALA may be converted to GLAand stearidonic acid [“STA”], respectively, by a Δ6 desaturase; then, aC_(18/20) elongase converts GLA to DGLA and/or STA to ETA.

Economical commercial production of EPA in a recombinant Yarrowia sp.host cell requires consideration of a variety of variables, includingthe EPA concentration [“EPA % TFAs”] and total lipid content [“TFAs %DCW”]. Furthermore, it is desirable to reduce the production ofintermediate fatty acids and byproduct fatty acids in the final oilproduct, in order to maximize production of the desired fatty acid,i.e., EPA.

Intermediate fatty acids are those fatty acids (e.g., oleic acid, LA,ALA, EDA, DGLA, ETA) that can be further converted to EPA by the actionof other metabolic pathway enzymes. In contrast, by-product fatty acids(e.g., sciadonic acid, juniperonic acid) refer to any fatty acidproduced that is neither EPA nor an intermediate fatty acid that can befurther converted to EPA.

U.S. Pat. Appl. Pub. No. 2009-0093543-A1 describes optimized strains ofrecombinant Yarrowia lipolytica having the ability to produce microbialoils comprising at least about 43.3 EPA % TFAs, with less than about23.6 LA % TFAs (an EPA:LA ratio of 1.83). The preferred strain wasY4305, whose maximum production was 55.6 EPA % TFAs, with an EPA:LAratio of 3.03. Generally, the EPA strains of U.S. Pat. Appl. Pub. No.2009-0093543-A1 comprised the following genes of the ω-3/ω-6 fatty acidbiosynthetic pathway:

-   -   a) at least one gene encoding Δ9 elongase; and,    -   b) at least one gene encoding Δ8 desaturase; and,    -   c) at least one gene encoding Δ5 desaturase; and,    -   d) at least one gene encoding Δ17 desaturase; and,    -   e) at least one gene encoding Δ2 desaturase; and,    -   f) at least one gene encoding C_(16/18) elongase; and,    -   g) optionally, at least one gene encoding diacylglycerol        cholinephosphotransferase (CPT1).        Examples of preferred genes having the enzymatic functionalities        described above are set forth in Table 3 (although these genes        are not intended to be limiting).

TABLE 3 Preferred Desaturases And Elongases For EPA Biosynthesis InYarrowia lipolytica Wildtype Codon-Optimized Mutant Co-pending PatentApplication Abbreviation and Abbreviation and Abbreviation and ORFOrganism References SEQ ID NO SEQ ID NO SEQ ID NO Δ9 Euglena gracillisU.S. Pat. No. 7,645,604 “EgD9e” “EgD9eS” — elongase (SEQ ID NOs: 43 (SEQID NOs: 45 and 44) and 46) Eutreptiella sp. U.S. Pat. No. 7,645,604“E389D9e” “E389D9eS” — CCMP389 (SEQ ID NOs: 47 (SEQ ID NOs: 49 and 48)and 50) Euglena U.S. Pat. Appl. Pub. No. 2008- “EaD9e”* “EaD9eS” —anabaena 0254522-A1; Intl. App. Pub. No. (SEQ ID NOs: 51 (SEQ ID NOs: 53UTEX 373 WO 2008/128241 and 52) and 54) Δ8 Euglena gracilis U.S. Pat.No. 7,256,033; “EgD8”* “EgD8S”* “EgD8M”* desaturase U.S. Pat. No.7,709,239 (SEQ ID NOs: 55 (SEQ ID NOs: 57 (SEQ ID NOs: 59 and 56) and58) and 60) Euglena U.S. Pat. Appl. Pub. No. 2008- “EaD8”* “EaD8S” —anabaena 0254521-A1; Intl. App. Pub. No. (SEQ ID NOs: 61 (SEQ ID NOs: 63UTEX 373 WO 2008/124194 and 62) and 64) Δ5 Euglena gracilis U.S. Pat.No. 7,678,560; U.S. Pat. “EgD5” “EgD5S” “EgD5M” (SEQ desaturase Pub. No.2010-0075386-A1 (SEQ ID NOs: 65 (SEQ ID NOs: 67 ID NOs: 69 and and 66)and 68) 70); “EgD5SM” (SEQ ID NOs: 71 and 72) Peridinium sp. U.S. Pat.No. 7,695,950; U.S. Pat. “RD5” “RD5S” — CCMP626 Pub. No. 2010-0075386-A1(SEQ ID NOs: 73 (SEQ ID NOs: 75 and 74) and 76) Euglena U.S. Pat. Appl.Pub. No. 2008- “EaD5”* “EaD5S”* “EaD5SM” anabaena 0274521-A1; U.S. Pat.Pub. No. (SEQ ID NOs: 77 (SEQ ID NOs: 79 (SEQ ID NOs: 81 UTEX 3732010-0075386-A1 and 78) and 80) and 82) Δ17 Phytophthora U.S. Pat. No.7,465,793 “PrD17” “PrD17S” — desaturase ramorum (SEQ ID NOs: 83 (SEQ IDNOs: 85 and 84) and 86) Pythium U.S. Pat. No. 7,556,949 “PaD17” “PaD17S”— aphanidematum (SEQ ID NOs: 87 (SEQ ID NOs: 89 and 88) and 90) Δ12Fusarium U.S. Pat. No. 7,504,259 “FmD12”* “FmD12S” — desaturasemoniliforme (SEQ ID NOs: 91 (SEQ ID NOs: 93 and 92) and 94) C_(16/18)Mortierella U.S. Pat. No. 7,470,532 “ELO3” “ME3S” — elongase alpina (SEQID NOs: 95 (SEQ ID NOs: 97 and 96) and 98) Diacyl- Yarrowia Intl. App.Pub. No. WO “YICPT” — — glycerol lipolytica 2006/052870 (SEQ ID NOs: 37choline- and 38) phospho- transferase *Notes: EaD9e was identified as“EaD9Elo1” in U.S. Pat. Appl. Pub. No. 2008-0254522-A1; EgD8 wasidentified as “Eg5” in U.S. Pat. No. 7,256,033; EgD8S was identified as“D8SF” in U.S. Pat. No. 7,256,033; EgD8M was identified as “EgD8S-23” inU.S. Pat. No. 7,709,239; EaD8 was identified as “EaD8Des3” in U.S. Pat.Appl. Pub. No. 2008-0254521-A1; EaD5 was identified as “EaD5Des1” inU.S. Pat. Appl. Pub. No. 2008-0274521-A1; and, FmD12 was identified as“Fm2” in U.S. Pat. No. 7,504,259.

Provided herein are optimized strains of recombinant Yarrowia lipolyticahaving the ability to produce improved microbial oils relative to thosestrains described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, based onthe EPA % TFAs and the ratio of EPA:LA. In addition to expressing genesof the ω-3/ω-6 fatty acid biosynthetic pathway as defined above and asdetailed in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, these improvedstrains are distinguished by:

-   -   1) comprising at least one multizyme, wherein said multizyme        comprises a polypeptide having at least one fatty acid Δ9        elongase linked to at least one fatty acid Δ8 desaturase [a        “DGLA synthase”];    -   2) optionally comprising at least one polynucleotide encoding an        enzyme selected from the group consisting of a malonyl CoA        synthetase or an acyl-CoA lysophospholipid acyltransferase        [“LPLAT”];    -   3) comprising at least one peroxisome biogenesis factor protein        whose expression has been down-regulated;    -   4) producing at least about 50 EPA % TFAs; and,    -   5) having a ratio of EPA:LA of at least about 3.1.

U.S. Pat. Appl. Pub. No. 2008-0254191-A1, and especially Examples 55 and56 which are hereby incorporated herein by reference, describes DGLAsynthases that possess improved enzymatic activity with respect to theirindividual Δ9 elongase and/or Δ8 desaturase counterparts, whenheterologously expressed in Yarrowia lipolytica. Particularly relevantto the disclosure herein, a linker sequence (i.e., SEQ ID NO:1[GAGPARPAGLPPATYYDSLAVMGS]) was used to fuse a Δ9 elongase (i.e.,EgD9eS, EaD9eS or E389D9eS) to a Δ8 desaturase (i.e., EgD8M or EaD8S),thereby creating EgD9eS/EgD8M (SEQ ID NOs:8 and 9), EaD9eS/EaD8S (SEQ IDNOs:10 and 11) and E389D9eS/EgD8M (SEQ ID NOs:12 and 13). Surprisingly,fusing the two independent enzymes together as one fusion proteinseparated by a linker region increased flux from LA to DGLA, suggestingthat the product of Δ9 elongase may be directly channeled as substrateof Δ8 desaturase in the fusion protein.

Table 4 below provides a summary of the improvements noted in conversionefficiency in U.S. Pat. Appl. Pub. No. 2008-0254191-A1, as a result ofthe gene fusion. Specifically, the number shown in bold text is thepercent improvement in elongase or desaturase activity, while thedetails shown in parentheses provide the elongase or desaturaseconversion efficiency in the gene fusion versus when the elongase ordesaturase conversion efficiency when the gene was expressed alone.

TABLE 4 Improvement In Δ9 Elongase And Δ8 Desaturase Conversion As AResult Of Gene Fusion Gene fusion Δ9 Improvement Δ8 ImprovementEgD9eS/EgD8M  5% 97% (SEQ ID NOs: 8 (21% versus 20% (73% versus 37% and9) conversion) conversion) EaD9eS/EaD8S 38% 32% (SEQ ID NOs: 10 (18%versus 13% (58% versus 41% and 11) conversion) conversion)E389D9eS/EgD8M 50% 89% (SEQ ID NOs: 12 (18% versus 12% (70% versus 37%and 13) conversion) conversion)

Based on the results described above, expression of at least one DGLAsynthase, such as the EgD9eS/EgD8M, EaD9eS/EaD8S and E389D9eS/EgD8M genefusions described above, is preferred in improved optimized strains ofrecombinant Yarrowia lipolytica having the ability to produce improvedEPA % TFAs. This gene fusion can be created using any combination ofpreferred Δ9 elongases and Δ8 desaturases suitable for expression in Y.lipolytica; and, the linker can be selected from the group consistingof: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 and SEQ ID NO:7.

Previous studies have determined that many of the genetic mutationsrelating to engineering production of PUFAs in Yarrowia lipolyticaresult in increased by production of malonates during the fermentation(malonates accounted for ˜45% of the total organic acids accumulated).Expression of a heterologous malonyl-CoA synthetase reversed this effectand resulted in substantially reduced by production of malonates.

More specifically, U.S. patent application Ser. No. 12/637,877 (filedDec. 15, 2009 and having Attorney Docket No. CL4323) describesgeneralized methods to avoid accumulation of organic acid (and inparticular, malonate) “byproducts” that cannot be further utilizedduring a fermentation, during production of a product. This avoidscarbon and energy waste within the organism, reduces the amount of baserequired to maintain an optimal pH range during the fermentationprocess, and reduces the amount of byproduct organic acids that requireneutralization within the fermentation waste steam.

Malonyl-CoA synthetase [EC 6.2.1.-] catalyzes the following enzymaticreaction: malonate+ATP+CoA→malonyl-CoA+AMP+pyrophosphate (PPi). Byconverting the byproduct (i.e., malonate) into malonyl-CoA, thissubstrate becomes available for use during the synthesis of fatty acidswithin the organism. Specifically, fatty acid synthesis can besummarized by the following equation (ignoring H⁺ and water):acetyl-CoA+7 malonyl-CoA+14 NADPH→palmitate+7 CO₂+14 NADP⁺+8 CoA.

A codon-optimized malonyl-CoA synthetase was created and expressed inYarrowia lipolytica in U.S. patent application Ser. No. 12/637,877.Specifically, the codon-optimized malonyl-CoA synthetase gene (“MCS”,SEQ ID NO:41) was designed based on the coding sequence of themalonyl-CoA synthetase gene from Rhizobium leguminosarum bv. viciae 3841(rMCS; SEQ ID NOs:39 and 40, corresponding to GenBank Accession No.YP_(—)766603). In addition to modification of the translation initiationsite, 233 by of the 1515 by coding region (including the stop codon)were modified (15.4%), 219 codons were optimized (43.4%), the GC contentwas reduced from 61.4% within the wild type gene (i.e., rMCS) to 55.6%within the synthetic gene (i.e., MCS) and the translation initiationcodon “ATG” was added in front of the rMCS gene (SEQ ID NO:39) sinceYarrowia cannot use the “GTG” codon for translation initiation. Thecodon-optimized MCS gene (SEQ ID NO:41) is 1518 by encoding a peptide of505 amino acids and a stop codon (SEQ ID NO:42).

Expression of MCS (SEQ ID NO:42) in Yarrowia lipolytica strain Y4305U,producing 49 EPA % TFAs, lowered the total amount of malonates (g/g DCW)˜94% without impacting either the fatty acid profile or the total lipidyield (TFAs % DCW).

Based on the results described above, expression of at least onemalonyl-CoA synthetase in improved optimized strains of recombinantYarrowia lipolytica is desirable, as a means to reduce generation ofunwanted byproducts and thereby decrease the cost of manufacture.Preferred malonyl-CoA synthetases are set forth as SEQ ID NOs:40 and 42,but these are should not be limiting to the disclosure herein. Oneskilled in the art could readily identify alternate heterologousmalonyl-CoA synthetases suitable for expression in Y. lipolytica.

Glycerophospholipids, the main component of biological membranes,contain a glycerol core with fatty acids attached as R groups at thesn-1 position and sn-2 position, and a polar head group joined at thesn-3 position via a phosphodiester bond. Table 5 below summarizes thesteps in the de novo biosynthetic pathway, originally described byKennedy and Weiss (J. Biol. Chem., 222:193-214 (1956)):

TABLE 5 General Reactions Of de Novo Glycerophospholipid Biosynthesissn-Glycerol-3-Phosphate Glycerol-3-phosphate acyltransferase (GPAT) →Lysophosphatidic Acid [E.C. 2.3.1.15] esterifies 1st acyl-CoA to sn-1(1-acyl-sn-glycerol 3- position of sn-glycerol 3-phosphate phosphate or“LPA”) LPA → Phosphatidic Acid Lysophosphatidic acid acyltransferase(LPAAT) (1,2-diacylglycerol [E.C. 2.3.1.51] esterifies 2^(nd) acyl-CoAto sn-2 phosphate or “PA”) position of LPA PA → 1,2-DiacylglycerolPhosphatidic acid phosphatase [E.C. 3.1.3.4] (“DAG”) removes a phosphatefrom PA; DAG can Or subsequently be converted to PA → CytidineDiphosphate phosphatidylcholines [“PC”], Diacylglycerolphosphatidylethanolamines [“PE”] or TAG (TAG (“CDP-DG”) synthesisrequires either a diacylglycerol acyltransferase (DGAT) [E.C. 2.3.1.20]or a phospholipid:diacylglycerol acyltransferase (PDAT) [E.C.2.3.1.158])CDP-diacylglycerol synthase [EC 2.7.7.41] causes condensation of PA andcytidine triphosphate, with elimination of pyrophosphate; CDP-DG cansubsequently be converted to phosphatidylglycerols [“PG”],phosphatidylinositols [“PI”], phosphatidylserines [“PS”] or cardiolipins[“CL”]

Following their de novo synthesis, glycerophospholipids can undergorapid turnover of their fatty acyl composition at the sn-2 position.This “remodeling”, or “acyl editing”, has been attributed to deacylationof the glycerophospholipid and subsequent reacylation of the resultinglysophospholipid.

In the Lands' cycle (Lands, W. E., J. Biol. Chem., 231:883-888 (1958)),remodeling occurs through the concerted action of: 1) a phospholipase,such as phospholipase A₂, that releases fatty acids from the sn-2position of phosphatidylcholine; and, 2) acyl-CoA:lysophospholipidacyltransferases [“LPLATs”], such as lysophosphatidylcholineacyltransferase [“LPCAT”] that reacylates the lysophosphatidylcholine[“LPC”] at the sn-2 position. Other glycerophospholipids can also beinvolved in the remodeling with their respective lysophospholipidacyltransferase activity, including LPLAT enzymes havinglysophosphatidylethanolamine acyltransferase [“LPEAT”] activity,lysophosphatidylserine acyltransferase [“LPLAT”] activity,lysophosphatidylglycerol acyltransferase [“LPGAT”] activity andlysophosphatidylinositol acyltransferase [“LPIAT”] activity. In allcases, LPLATs are responsible for removing acyl-CoA fatty acids from thecellular acyl-CoA pool and acylating various lysophospholipid substratesat the sn-2 position in the phospholipid pool. Finally, LPLATs alsoinclude LPAAT enzymes that are involved in the de novo biosynthesis ofPA from LPA.

In other cases, this sn-2 position remodeling has been attributed to theforward and reverse reactions of enzymes having LPCAT activity (StymneS, and A. K. Stobart, Biochem. J., 223(2):305-314 (1984)).

Several recent reviews by Shindou et al. provide an overview ofglycerophospholipid biosynthesis and the role of LPLATs (J. Biol. Chem.,284(1):1-5 (2009); J. Lipid Res., 50:S46-S51 (2009)). And, numerousLPLATs have been reported in public and patent literature, based on thepresence of conserved motifs.

More specifically, a variety of LPLAT motifs have been proposed, withslight variation based on the specific species that are included inanalyzed alignments. For example, Shindou et al. (Biochem. Biophys. Res.Comm., 383:320-325 (2009)) proposed the following membrane boundO-acyltransferase [“MBOAT”] family motifs to be important for LPLATactivity, based on alignment of sequences from Homo sapiens, Gallusgallus, Danio rerio and Caenorhabditis elegans: WD, WHGxxxGYxxxF (SEQ IDNO:99), YxxxxF (SEQ ID NO:100) and

YxxxYFxxH (SEQ ID NO:101). Of these, the WD, WHGxxxGYxxxF and YxxxxFmotifs are present in ScAle (SEQ ID NO:15) and YIAle1 (SEQ ID NO:17),but the YxxxYFxxH motif is not. Alternate non-plant motifs for Ale1homologs are also described in U.S. Pat. Appl. Pub. No. 2008-0145867-A1;specifically, these include: M-[V/I]-[L/I]-xxK-[L/V/1]-xxxxxxDG (SEQ IDNO:102), RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:103),EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:21) and SAxWHGxxPGYxx-[T/F]-F (SEQ IDNO:104).

Similarly, Lewin, T. W. et al. (Biochemistry, 38:5764-5771 (1999) andYamashita et al. (Biochim. Biophys. Acta, 1771:1202-1215 (2007))proposed the following 1-acyl-sn-glycerol-3-phosphate acyltransferase[“LPAAT”] family motifs to be important for LPLAT activity, based onalignment of sequences from bacteria, yeast, nematodes and mammals:NHxxxxD (SEQ ID NO:33), GxxFI-[D/R]-R (SEQ ID NO:105), EGTR (SEQ IDNO:34) and either [V/I]-[P/X]-[I/V/L]-[I/V]-P-[V/I] (SEQ ID NO:106) orIVPIVM (SEQ ID NO:107). The NHxxxxD and EGTR motifs are present inMaLPAAT1 (SEQ ID NO:29), YILPAAT1 (SEQ ID NO:31) and CeLPCAT (SEQ IDNO:25), but the other LPAAT family motifs are not.

Based on publicly available Ale1, LPCAT and LPAAT protein sequences,including those described herein, LPLATs for inclusion in the improvedoptimized strains of recombinant Yarrowia lipolytica herein possesseither MBOAT family motifs selected from the group consisting of:M(V/I)LxxKL (SEQ ID NO:18), RxKYYxxW (SEQ ID NO:19), SAxWHG (SEQ IDNO:20) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:21) or1-acyl-sn-glycerol-3-phosphate acyltransferase family motifs selectedfrom the group consisting of: NHxxxxD (SEQ ID NO:33) and EGTR (SEQ IDNO:34).

The effect of LPLATs on PUFA production has been contemplated, sincefatty acid biosynthesis requires rapid exchange of acyl groups betweenthe acyl-CoA pool and the phospholipid pool. Specifically, desaturationsoccur mainly at the sn-2 position of phospholipids, while elongationoccurs in the acyl-CoA pool.

More specifically, it has been previously hypothesized that LPCATs couldbe important in the accumulation of EPA in the TAG fraction of Yarrowialipolytica (U.S. Pat. Appl. Pub. No. 2006-0115881-A1). As describedtherein, this hypothesis was based on the following studies: 1) StymneS, and A. K. Stobart (Biochem. J., 223(2):305-314 (1984)), whohypothesized that the exchange between the acyl-CoA pool and PC pool maybe attributed to the forward and backward reaction of LPCAT; 2)Domergue, F. et al. (J. Bio. Chem., 278:35115 (2003)), who suggestedthat accumulation of GLA at the sn-2 position of PC and the inability toefficiently synthesize ARA in yeast was a result of the elongation stepinvolved in PUFA biosynthesis occurring within the acyl-CoA pool, whilethe Δ5 and Δ6 desaturation steps occurred predominantly at the sn-2position of PC; 3) Abbadi, A. et al. (The Plant Cell, 16:2734-2748(2004)), who suggested that LPCAT plays a critical role in thesuccessful reconstitution of a Δ6 desaturase/Δ6 elongase pathway, basedon analysis on the constraints of PUFA accumulation in transgenicoilseed plants; and, 4) the work of Renz, A. et al. in Intl. App.Publications No. WO 2004/076617 A2 and No. WO 2004/087902 A2.

More specifically, Intl. App. Pub. No. WO 2004/076617 A2 describes theisolation of a LPCAT from Caenorhabditis elegans (clone T06E8.1)[“CeLPCAT”] and reports increase in the efficiency of Δ6 desaturationand Δ6 elongation, as well as an increase in biosynthesis of thelong-chain PUFAs eicosadienoic acid [“EDA”; 20:2] and eicosatetraenoicacid [“ETA”; 20:4], respectively, when the LPCAT was expressed in anengineered strain of Saccharomyces cerevisiae that was fed exogenous18:2 or α-linolenic [“ALA”; 18:3] fatty acids, respectively.

Intl. App. Pub. No. WO 2004/087902 A2 (Example 16) describes theisolation of Mortierella alpina LPAAT-like proteins (encoded by proteinshaving 417 amino acids in length or 389 amino acids in length,respectively, that are identical except for an N-terminal extension of28 amino acid residues) and reports expression of one of these proteinsusing similar methods to those of Intl. App. Pub. No. WO 2004/076617 A2,which results in similar improvements in EDA and ETA biosynthesis.

Both Intl. App. Publications No. WO 2004/076617 and No. WO 2004/087902teach that the improvement in EDA and ETA biosynthesis is due toreversible LPCAT activity in CeLPLAT and some LPAAT-like proteins,although not all LPAAT-like proteins have LPCAT activity. Furthermore,Renz, A. et al. concluded that LPCAT allowed efficient and continuousexchange of the newly synthesized fatty acids between phospholipids andthe acyl-CoA pool, since desaturases catalyze the introduction of doublebonds in PC-coupled fatty acids while elongases exclusively catalyze theelongation of CoA esterified fatty acids (acyl-CoAs).

Numerous other references generally describe benefits of co-expressingLPLATs with PUFA biosynthetic genes, to increase the amount of a desiredfatty acid in the oil of a transgenic organism, increase total oilcontent or selectively increase the content of desired fatty acids(e.g., Intl. App. Publications No. WO 2006/069936, No. WO 2006/052870,No. WO 2009/001315, No. WO 2009/014140).

Herein (and in Applicant's Assignee's co-filed U.S. Provisional PatentApplication No. 61/187,359, filed Jun. 16, 2009, having Attorney DocketNo. CL4361 USPRV, incorporated by reference in its entirety), it isdemonstrated that LPAAT and LPCAT are indeed important in theaccumulation of EPA in the TAG fraction of Yarrowia lipolytica.Specifically, it was found that over-expression of LPLATs can result inan improvement in the Δ9 elongase conversion efficiency. As previouslydefined, conversion efficiency is a term that refers to the efficiencyby which a particular enzyme, such as a Δ9 elongase, can convertsubstrate (e.g., LA) to product (e.g., EDA). Thus, in a strainengineered to produce EPA, improvement in Δ9 elongase conversionefficiency was demonstrated to result in increased EPA % TFAs and/or EPA% DCW.

These results, and additional supporting work, are the cornerstone ofthe following claimed method for improving C₁₈ to C₂₀ elongationconversion efficiency in a LC-PUFA-producing recombinant oleaginousmicrobial host cell, wherein said method comprises:

a) introducing into said LC-PUFA-producing recombinant host cell atleast one isolated polynucleotide encoding a polypeptide having at leastacyl-CoA:lysophospholipid acyltransferase activity wherein thepolypeptide is selected from the group consisting of:

-   -   (i) a polypeptide having at least 45% amino acid identity, based        on the Clustal W method of alignment, when compared to an amino        acid sequence selected from the group consisting of SEQ ID NO:15        (ScAle1) and SEQ ID NO:17 (YIAle1);    -   (ii) a polypeptide having at least one membrane bound        O-acyltransferase protein family motif selected from the group        consisting of: M(V/I)LxxKL (SEQ ID NO:18), RxKYYxxW (SEQ ID        NO:19), SAxWHG (SEQ ID NO:20) and EX₁₁WNX₂-[T/V]-X₂W (SEQ ID        NO:21);    -   (iii) a polypeptide having at least 90% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence as set forth in SEQ ID NO:25 (CeLPCAT);    -   (iv) a polypeptide having at least 43.9% amino acid identity,        based on the Clustal W method of alignment, when compared to an        amino acid sequence selected from the group consisting of SEQ ID        NO:29 (MaLPAAT1), SEQ ID NO:31 (YILPAAT1) and SEQ ID NO:32        (ScLPAAT1); and,    -   (v) a polypeptide having at least one        1-acyl-sn-glycerol-3-phosphate acyltransferase protein family        motif selected from the group consisting of: NHxxxxD (SEQ ID        NO:33) and EGTR (SEQ ID NO:34);

wherein the at least one isolated polynucleotide encoding a polypeptidehaving at least acylCoA:lysophospholipid acyltransferase activity isoperably linked to at least one regulatory sequence, said regulatorysequence being the same or different; and,

b) growing the oleaginous microbial host cell;

wherein the C₁₈ to C₂₀ elongation conversion efficiency of theoleaginous microbial host cell is increased relative to the control hostcell.

Preferably, the polynucleotide encoding a polypeptide having at leastacyl-CoA:lysophospholipid acyltransferase activity is stably integratedand the an increase in C₁₈ to C₂₀ elongation conversion is at leastabout 4%,

More preferred, the increase in C₁₈ to C₂₀ elongation conversionefficiency is at least about 4-10%, more preferred at least about10-20%, more preferred at least about 20-40%, and most preferred atleast about 40-60% or greater in at least one LC-PUFA-producingoleaginous microbial host cell when compared to the control host cell.

Based on the improvement in C₁₈ to C₂₀ elongation conversion efficiencydescribed above, optimized strains of recombinant Yarrowia lipolyticahaving the ability to produce improved EPA % TFAs, relative to thosestrains described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, willoptionally comprise at least one acyl-CoA lysophospholipidacyltransferase [“LPLAT”] as defined in the methods described above. Inpreferred embodiments, the amino acid sequence of the LPLAT is selectedfrom the group consisting of: SEQ ID NO:15 (ScAle1), SEQ ID NO:16(YIAle1), SEQ ID NO:25 (CeLPCAT), SEQ ID NO:29 (MaLPAAT1), SEQ ID NO:31(YILPAAT1) and SEQ ID NO:32 (ScLPAAT1).

U.S. Pat. Appl. Pub. No. 2009-0093543-A1 describes a variety ofknockouts useful in recombinant Yarrowia sp., including those useful forselection of transformants, those that diminish fatty acid degradationand TAG degradation and those that appear to result in a phenotypically“neutral” mutation (wherein the Yarrowia host cell seems unaffected).Most preferred, however, are those gene knockouts (e.g., diacylglycerolacyltransferase gene knockouts, peroxisome biogenesis factor protein[“PEX”] gene knockouts) that result in increases in the concentration ofEPA relative to the total fatty acids [“EPA % TFAs”].

More specifically, U.S. Pat. Appl. Pub. No. 2009-0093543-A1 contemplatesthat in some preferred recombinant Yarrowia production hosts, the hostis devoid of a native gene encoding a peroxisome biogenesis factorprotein selected from the group consisting of: Pex1p (SEQ ID NO:108),Pex2p (SEQ ID NO:109), Pex3p (SEQ ID NO:110), Pex3 Bp (SEQ ID NO:111),Pex4p (SEQ ID NO:112), Pex5p (SEQ ID NO:113), Pex6p (SEQ ID NO:114),Pex7p (SEQ ID NO:115), Pex8p (SEQ ID NO:116), Pex10p (SEQ ID NO:117),Pex12p (SEQ ID NO:118), Pex13p (SEQ ID NO:119), Pex14p (SEQ ID NO:120),Pex16p (SEQ ID NO:121), Pex17p (SEQ ID NO:122), Pex19p (SEQ ID NO:123),Pex20p (SEQ ID NO:124), Pex22p (SEQ ID NO:125) and Pex26p (SEQ IDNO:126). More preferred disrupted peroxisome biogenesis factor proteinsare Pex2p, Pex3p, Pex10p, Pex12p and Pex16p, although data is providedonly concerning Pex10p.

Intl. App. Pub. No. WO 2009/046248 confirms and expands the hypothesesand studies presented in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, bycomparing ΔPex16, ΔPex3 and ΔPex10 strains of Yarrowia lipolytica.Results therein demonstrated that Pex10 disruption was responsible for a3.3 fold increase in EPA % TFAs and a 1.7 fold increase in the amount ofC₂₀ PUFAs relative to the non-disrupted strain engineered for EPAproduction. Similarly, a 1.65 fold increase in DGLA

TFAs and a 1.3 fold increase in C₂₀ PUFAs % TFAs was observed in aΔPex16 strain engineered for DGLA production. A 2.0 fold increase inDGLA % TFAs and a 1.7 fold increase in C₂₀ PUFAs % TFAs was observed ina ΔPex3 strain engineered for DGLA production.

These results, and additional supporting work, are the cornerstone ofthe following claimed method for increasing the weight percent of onePUFA or a combination of PUFAs relative to the weight percent of totalfatty acids in an oleaginous eukaryotic organism having a total lipidcontent, a total lipid fraction and an oil fraction, said methodcomprising:

-   -   a) providing an oleaginous eukaryotic organism comprising a        disruption in a native gene encoding a peroxisome biogenesis        factor protein, which creates a PEX-disruption organism; and        genes encoding a functional PUFA biosynthetic pathway; and,    -   b) growing the PEX-disrupted organism under conditions wherein        the weight percent of at least one PUFA is increased in the        total lipid fraction and in the oil fraction relative to the        weight percent of the total fatty acids, when compared with        those weight percents in an oleaginous eukaryotic organism whose        native peroxisome biogenesis factor protein has not been        disrupted.        The amount of PUFAs that increases as a percent of total fatty        acids can be: 1) the PUFA that is the desired end product of a        functional PUFA biosynthetic pathway, as opposed to PUFA        intermediates or by-products; 2) C₂₀ to C₂₂ PUFAs; and/or, 3)        total PUFAs.

In addition to the increase in the weight percent of one or acombination of PUFAs relative to the weight percent of the total fattyacids, in some cases, the total lipid content (TFA % DCW) of the cellmay be increased or decreased. What this means is that regardless ofwhether the disruption in the PEX gene causes the amount of total lipidsin the PEX-disrupted cell to increase or decrease, the disruption alwayscauses the weight percent of a PUFA or of a combination of PUFAs toincrease.

Based on the above, optimized strains of recombinant Yarrowia lipolyticahaving the ability to produce improved EPA % TFAs, relative to thosestrains described in U.S. Pat. Appl. Pub. No. 2009-0093543-A1, willcomprise at least one peroxisome biogenesis factor protein whoseexpression has been down-regulated (i.e., thereby producing aPEX-disrupted organism). In preferred strains, the down-regulatedperoxisome biogenesis factor protein is Pex3p (SEQ ID NO:110), Pex10p(SEQ ID NO:117), or Pex16p (SEQ ID NO:121).

Although numerous techniques are available to one of skill in the art toachieve disruption of a native Yarrowia gene, generally the endogenousactivity of a particular gene can be reduced or eliminated by thefollowing techniques, for example: 1) disrupting the gene throughinsertion, substitution and/or deletion of all or part of the targetgene; or, 2) manipulating the regulatory sequences controlling theexpression of the protein. Both of these techniques are discussed inU.S. Pat. Appl. Pub. No. 2009-0093543-A1, as well as Intl. App. Pub. No.WO 2009/046248. One skilled in the art would appreciate that these andother methods are well described in the existing literature and are notlimiting to the methods, host cells, and products described herein. Oneskilled in the art will also appreciate the most appropriate techniquefor use with any particular oleaginous yeast.

The optimized strains will produce at least about 40-50 EPA % TFAs,preferably at least about 50-55 EPA % TFAs, more preferably at leastabout 55-60 EPA % TFAs, more preferably at least 60-70 EPA % TFAs, andmost preferably at least about 70-80 EPA % TFAs.

As will be clear to one of skill in the art, a multitude of differentoptimized Yarrowia strains producing at least about 50 EPA % TFAs couldbe engineered using the methodologies described herein. Selection of apreferred strain for commercial purposes will therefore also considerthe total lipid content of the engineered strain, since both theconcentration of EPA as a percent of the total fatty acids [“EPA %TFAs”] and total lipid content [“TFAs % DCW”] affect the cellularcontent of EPA as a percent of the dry cell weight [“EPA % DCW”]. Thatis, EPA % DCW is calculated as: (EPA % TFAs)*(TFA % DCW)]/100. Forexample, a strain producing 50 EPA % TFAs and having 24 TFAs % DCW, astrain producing 55 EPA % TFAs and having 21.82 TFAs % DCW, a strainproducing 60 EPA % TFAs and having 20 TFAs % DCW, a strain producing 65EPA % TFAs and having 18.46 TFAs % DCW and a strain producing 70 EPA %TFAs and having 17.14 TFAs % DCW all produce 12 EPA % DCW. In preferredembodiments, the improved optimized strain of Yarrowia lipolytica willproduce at least about 10-12 EPA % DCW, preferably at least about 12-14EPA % DCW, more preferably at least about 14-16 EPA % DCW, morepreferably at least about 16-18 EPA % DCW, more preferably at leastabout 18-20 EPA % DCW, more preferably at least about 20-22 EPA DCW,more preferably at least about 22-24 EPA % DCW, and most preferably atleast about 24-26 EPA % DCW.

In addition to possessing at least about 50 EPA % TFAs, the lipidprofile within the improved optimized strain of Yarrowia lipolytica, orwithin extracted or unconcentrated oil therefrom, will have a ratio ofEPA % TFAs to LA % TFAs of at least about 3.1. As demonstrated in U.S.Pat. Appl. Pub. No. 2009-0093543-A1 (Table 23), EPA, LA and oleic acidconstituted approximately 76-80% of the fatty acids present in the lipidprofile of a strain of Y. lipolytica producing greater than 40 EPA %TFAs. Of this, LA % TFAs was ca. three-fold greater than oleic acid %TFAs. Based on these observations, one of skill in the art willappreciate that minimizing the concentration of the intermediate fattyacid, LA (resulting in increased ratios of EPA:LA), will result ingreater “pushing” of the carbon through the PUFA biosynthetic pathwayand permit increased synthesis of EPA. In preferred embodiments, theratio of EPA:LA will be at least about 3.1-3.5, more preferably at leastabout 3.5-4.5, more preferably at least about 4.5-5.5, and mostpreferably at least about 5.5-6.5.

Lipids produced by the improved optimized recombinant Y. lipolyticastrains described herein will also be distinguished as having less thanabout 0.5% GLA or DHA (when measured by GC analysis using equipmenthaving a detectable level down to about 0.1%) and having a saturatedfatty acid content of less than about 8%. This low percent of saturatedfatty acids (i.e., 16:0 and 18:0) results in substantial health benefitsto humans and animals.

Microbial expression systems and expression vectors containingregulatory sequences that direct high-level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes encoding the preferred desaturase,elongase, CPT1, DGLA synthase, malonyl CoA synthetase and acyl-CoAlysophospholipid acyltransferase proteins. These chimeric genes couldthen be introduced into Yarrowia lipolytica using standard methods oftransformation to provide high-level expression of the encoded enzymes.

Vectors (e.g., constructs, plasmids) and DNA expression cassettes usefulfor the transformation of Yarrowia host cells are well known in the art.The specific choice of sequences present in the construct is dependentupon the desired expression products, the nature of the host cell, andthe proposed means of separating transformed cells versusnon-transformed cells. Typically, however, the vector contains at leastone expression cassette, a selectable marker and sequences allowingautonomous replication or chromosomal integration. Suitable expressioncassettes typically comprise a region 5′ of the gene that controlstranscriptional initiation (e.g., a promoter), the gene coding sequence,and a region 3′ of the DNA fragment that controls transcriptionaltermination (i.e., a terminator). It is most preferred when both controlregions are derived from genes from the transformed host cell, althoughthey need not be derived from genes native to the production host (e.g.,Yarrowia lipolytica).

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other constructs to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct can be experimentally determinedso that all introduced genes are expressed at the necessary levels toprovide for synthesis of the desired products.

Constructs or vectors comprising the gene(s) of interest may beintroduced into a host cell such as Yarrowia by any standard technique.These techniques include transformation (e.g., lithium acetatetransformation [Methods in Enzymology, 194:186-187 (1991)]), bolisticimpact, electroporation, microinjection, or any other method thatintroduces the gene(s) of interest into the host cell. More preferredherein for Yarrowia lipolytica are integration techniques based onlinearized fragments of DNA, as described in U.S. Pat. No. 4,880,741 andU.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl. Microbiol.Biotechnol., 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) is referred toherein as “transformed”, “transformant” or “recombinant”. Thetransformed host will have at least one copy of the expression cassetteand may have two or more, depending upon whether the expression cassetteis integrated into the genome or is present on an extrachromosomalelement having multiple copy numbers. The transformed host cell can beidentified by various selection techniques, as described in U.S. Pat.No. 7,238,482 and U.S. Pat. No. 7,259,255.

Preferred selection methods for use herein are resistance to kanamycin,hygromycin and the amino glycoside G418, as well as ability to grow onmedia lacking uracil, leucine, lysine, tryptophan or histidine. Inalternate embodiments, 5-fluoroorotic acid (5-fluorouracil-6-carboxylicacid monohydrate; “5-FOA”) is used for selection of yeast Ura− mutants(U.S. Pat. Appl. Pub. No. 2009-0093543-A1), or a native acetohydroxyacidsynthase (or acetolactate synthase; E.G. 4.1.3.18) that confers sulfonylurea herbicide resistance (Intl. App. Pub. No. WO 2006/052870) isutilized for selection of transformants. A unique method of “recycling”a pair of preferred selection markers for their use in multiplesequential transformations, by use of site-specific recombinase systems,is also taught in U.S. Pat. Appl. Pub. No. 2009-0093543-A1.

As is well known to one of skill in the art, merely inserting a gene(e.g., a desaturase, elongase, CPT1, DGLA synthase, malonyl CoAsynthetase, acyl-CoA lysophospholipid acyltransferase) into a cloningvector does not ensure its expression at the desired rate,concentration, amount, etc. It may be desirable to manipulate a numberof different genetic elements that control aspects of transcription, RNAstability, translation, protein stability and protein location, oxygenlimitation and secretion from the host cell. More specifically, geneexpression may be controlled by altering the following: the nature ofthe relevant transcriptional promoter and terminator sequences; thenumber of copies of the cloned gene; whether the gene is plasmid-borneor integrated into the genome of the host cell; the final cellularlocation of the synthesized foreign protein; the efficiency oftranslation in the host organism; the intrinsic stability of the clonedgene protein within the host cell; and, the codon usage within thecloned gene, such that its frequency approaches the frequency ofpreferred codon usage of the host cell. Several of these methods ofoverexpression will be discussed below, and are useful in recombinantYarrowia host cells as a means to overexpress e.g., desaturases,elongases, CPT1 proteins, DGLA synthases, malonyl CoA synthetases andacyl-CoA lysophospholipid acyltransferases.

Expression of the desired gene(s) can be increased at thetranscriptional level through the use of a stronger promoter (eitherregulated or constitutive) to cause increased expression, byremoving/deleting destabilizing sequences from either the mRNA or theencoded protein, or by adding stabilizing sequences to the mRNA (U.S.Pat. No. 4,910,141).

Transcription initiation control regions or promoters which are usefulto drive expression of heterologous genes or portions thereof inYarrowia host cells are numerous and known to those skilled in the art.Expression can be accomplished in an induced or constitutive fashion.Induced expression can be accomplished by inducing the activity of aregulatable promoter operably linked to the gene of interest, whileconstitutive expression can be achieved by the use of a constitutivepromoter operably linked to the gene of interest. Virtually any promoter(i.e., native, synthetic, or chimeric) capable of directing expressionof desaturase, elongase, CPT1, DGLA synthase, malonyl CoA synthetase andacyl-CoA lysophospholipid acyltransferase genes in Yarrowia will besuitable, although transcriptional and translational regions from thehost species are particularly useful.

In general, the termination region can be derived from the 3′ region ofthe gene from which the initiation region was obtained or from adifferent gene. A large number of termination regions are known andfunction satisfactorily in a variety of hosts when utilized both in thesame and different genera and species from which they were derived. Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene. The 3′-region can alsobe synthetic, as one of skill in the art can utilize availableinformation to design and synthesize a 3′-region sequence that functionsas a transcription terminator. A termination site may be unnecessary,but it is highly preferred.

Although not intended to be limiting, preferred promoter regions andtermination regions useful in the disclosure herein are those taught inU.S. Pat. Pub. No. 2009-0093543-A1.

Additional copies (i.e., more than one copy) of the PUFA biosyntheticpathway desaturase, elongase and DGLA synthase genes and/or CPT1,malonyl CoA synthetase and acyl-CoA lysophospholipid acyltransferasegenes may be introduced into Yarrowia lipolytica to thereby increase EPAproduction and accumulation. Specifically, additional copies of genesmay be cloned within a single expression construct; and/or, additionalcopies of the cloned gene(s) may be introduced into the host cell byincreasing the plasmid copy number or by multiple integration of thecloned gene into the genome (infra).

It is important to note that the when preparing optimized strains of Y.lipolytica according to the methodology herein, copies of variousdesaturases, elongases, CPT1 s, DGLA synthases, malonyl CoA synthetasesand acyl-CoA lysophospholipid acyltransferases are often referred to.If, for example, 2 copies of a Δ9 elongase are required, this can referto: 1) two copies of an identical coding sequence for a particular Δ9elongase isolated from a single species; or, 2) one coding sequence fora Δ9 elongase isolated from a species “A” and one coding sequence for aΔ9 elongase isolated from a species “B”, thus collectively resulting intwo Δ9 elongases.

In general, once a DNA cassette (e.g., comprising a chimeric genecomprising a promoter, ORF and terminator) suitable for expression in anoleaginous yeast has been obtained, it is either placed in a plasmidvector capable of autonomous replication in a host cell or directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination with the host locus.Although not relied on herein, all or some of the transcriptional andtranslational regulatory regions can be provided by the endogenous locuswhere constructs are targeted to an endogenous locus.

The preferred method of expressing genes in Yarrowia lipolytica is byintegration of a linear DNA fragment into the genome of the host.Integration into multiple locations within the genome can beparticularly useful when high level expression of genes are desired.Preferred loci include those taught in U.S. Pat. Pub. No.2009-0093543-A1.

Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability of anintegrated DNA fragment in Yarrowia lipolytica is dependent on theindividual transformants, the recipient strain and the targetingplatform used. Thus, the skilled artisan will recognize that multipletransformants must be screened in order to obtain a strain displayingthe desired expression level and pattern. Such screening may beaccomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol.,98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J.Chromatogr. Biomed. Appl., 618 (1-2):133-145 (1993)), Western analysisof protein expression, phenotypic analysis or GC analysis of the PUFAproducts.

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric genes (e.g., encoding desaturases,elongases, CPT1, DGLA synthases, malonyl CoA synthetases, acyl-CoAlysophospholipid acyltransferases) and produce the greatest and the mosteconomical yield of EPA. In general, media conditions may be optimizedby modifying the type and amount of carbon source, the type and amountof nitrogen source, the carbon-to-nitrogen ratio, the amount ofdifferent mineral ions, the oxygen level, growth temperature, pH, lengthof the biomass production phase, length of the oil accumulation phaseand the time and method of cell harvest. Yarrowia lipolytica aregenerally grown in a complex media such as yeastextract-peptone-dextrose broth [“YPD”] or a defined minimal media thatlacks a component necessary for growth and thereby forces selection ofthe desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media for the methods and host cells described herein mustcontain a suitable carbon source, such as are taught in U.S. Pat. No.7,238,482 and U.S. patent application Ser. No. 12/641,929 (filed Dec.19, 2009). Although it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing sources, preferred carbon sources are sugars, glyceroland/or fatty acids. Most preferred is glucose, sucrose, invert sucrose,fructose and/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the highEPA-producing oleaginous yeast and the promotion of the enzymaticpathways for EPA production. Particular attention is given to severalmetal ions, such as Fe⁺², Cu⁺², Mn⁺², Co+², Zn⁺² and Mg⁺², that promotesynthesis of lipids and PUFAs (Nakahara, T. et al., Ind. Appl. SingleCell Oils, D. J. Kyle and R. Colin, eds. pp 61-97 (1992)).

Preferred growth media for the methods and host cells described hereinare common commercially prepared media, such as Yeast Nitrogen Base(DIFCO Laboratories, Detroit, Mich.). Other defined or synthetic growthmedia may also be used and the appropriate medium for growth of Yarrowialipolytica will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.5 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of EPA in Yarrowia lipolytica. This approach is described inU.S. Pat. No. 7,238,482, as are various suitable fermentation processdesigns (i.e., batch, fed-batch and continuous) and considerationsduring growth.

In some aspects herein, the primary product is oleaginous yeast biomass.As such, isolation and purification of the EPA-containing oils from thebiomass may not be necessary (i.e., wherein the whole cell biomass isthe product).

However, certain end uses and/or product forms may require partialand/or complete isolation/purification of the EPA-containing oil fromthe biomass, to result in partially purified biomass, purified oil,and/or purified EPA. PUFAs, including EPA, may be found in the hostmicroorganism (e.g., Yarrowia) as free fatty acids or in esterifiedforms such as acylglycerols, phospholipids, sulfolipids or glycolipids.These fatty acids may be extracted from the host cell through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology, 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh and0. Ward (Adv. Appl. Microbiol., 45:271-312 (1997)).

In general, means for the purification of EPA and other PUFAs fromYarrowia biomass may include extraction (e.g., U.S. Pat. No. 6,797,303and U.S. Pat. No. 5,648,564) with organic solvents, sonication,supercritical fluid extraction (e.g., using carbon dioxide),saponification and physical means such as presses, bead beaters, orcombinations thereof. One is referred to the teachings of U.S. Pat. No.7,238,482 for additional details.

Oils containing EPA that have been refined and/or purified can behydrogenated, to thereby result in fats with various melting propertiesand textures. Many processed fats, including spreads, confectionaryfats, hard butters, margarines, baking shortenings, etc., requirevarying degrees of solidity at room temperature and can only be producedthrough alteration of the source oil's physical properties. This is mostcommonly achieved through catalytic hydrogenation (see Intl. App. Pub.No. WO 2006/052870 for additional details and references).

Food products, infant formulas, functional foods, medical foods, medicalnutritionals, dietary supplements, pharmaceutical compositions, animalfeeds, and personal care products comprising oleaginous yeast biomasscomprising EPA are provided herein. Similarly, also provided are foodproducts, infant formulas, functional foods, medical foods, medicalnutritionals, dietary supplements, pharmaceutical compositions, animalfeeds, and personal care products comprising EPA or microbial oilcomprising EPA isolated from the recombinant oleaginous yeast biomass.

One of skill in the art of processing and formulation will understandhow the amount and composition of the biomass, partially purifiedbiomass, purified oil, and/or purified EPA may be added to a particularproduct according to target species and/or end use. More specifically,an “effective” amount should be incorporated into a product formulation,although this amount will depend on the food or feed product, the dietthat the product is intended to supplement or the medical condition thatthe medical food or medical nutritional is intended to correct or treat.Most desirably, the effective amount of EPA will be sufficient toprovide the desirable health characteristics associated with ω-3/ω-6PUFA consumption. Typically, the amount of EPA incorporated into theproduct takes into account losses associated with processing conditions,typical handling and storage conditions, the stability of EPA in theproduct, and the bioavailability/bioabsorption efficiency with thetarget species, to name a few.

One of skill in the art of processing and formulation will be familiarwith processes to concentrate the oil produced from the recombinantYarrowia production host cells described herein, to thereby increase theconcentration of EPA in the total lipid fraction such that it comprisesat least about 60%, at least about 70%, at least about 80% or even atleast about 90% EPA. Means to blend the purified oils described hereinwith other purified fatty acids (e.g., LA, GLA, EDA, DGLA, ARA, DTA,DPAn-6, ALA, STA, ETrA, ETA, DPA and DHA), or oils containing alternatefatty acids in preferred concentrations, are also well known to one ofskill in the art. These techniques readily permit the creation of an oilcomprising a uniquely tailored fatty acid profile.

Personal Care Products: Within the context of personal care products,ω-3 fatty acids have particular application in skin formulations wherethey may be used to enhance the skin conditioning effect. The skilledperson will understand how to provide an effective amount of therelevant ω-3 fatty acid(s) or oil comprising the same to a skin carecomposition. In addition to the PUFA oil or ω-3 fatty acid, the skincare composition may further comprise a cosmetically acceptable mediumfor skin care compositions, examples of which are described by Philippeet al. in U.S. Pat. No. 6,280,747. For example, the cosmeticallyacceptable medium may be an anhydrous composition containing a fattysubstance in a proportion generally from about 10% to about 90% byweight relative to the total weight of the composition, where the fattyphase contains at least one liquid, solid or semi-solid fatty substance.The fatty substance includes, but is not limited to, oils, waxes, gums,and so-called pasty fatty substances. Alternatively, the compositionsmay be in the form of a stable dispersion such as a water-in-oil oroil-in-water emulsion. Additionally, the compositions may contain one ormore conventional cosmetic or dermatological additives or adjuvantsincluding, but not limited to, antioxidants, preserving agents, fillers,surfactants, UVA and/or UVB sunscreens, fragrances, thickeners, wettingagents, anionic or nonionic or amphoteric polymers, and dyes.

Foodstuffs: The market place currently supports a large variety of foodand feed products, incorporating ω-3 and/or ω-6 fatty acids(particularly LA, GLA, ARA, EPA, DPA and DHA). It is contemplated thatthe yeast biomass, partially purified biomass, purified oil, and/orpurified EPA described herein will function in food products to impartthe health benefits of current formulations.

Food products will include, but not be limited to: food analogs, drinks,meat products, cereal products, baked foods, snack foods and dairyproducts.

Food analogs can be made using processes well known to those skilled inthe art. There can be mentioned meat analogs, cheese analogs, milkanalogs and the like. Meat analogs made from soybeans contain soyprotein or tofu and other ingredients mixed together to simulate variouskinds of meats. These meat alternatives are sold as frozen, canned ordried foods. Usually, they can be used the same way as the foods theyreplace. Examples of meat analogs include, but are not limited to: hamanalogs, sausage analogs, bacon analogs, and the like.

Food analogs can be classified as imitation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to:imitation milks and nondairy frozen desserts (e.g., those made fromsoybeans and/or soy protein products).

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processed meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice. Examples of cereal food products include, but are not limitedto: whole grain, crushed grain, grits, flour, bran, germ, breakfastcereals, extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and which has been baked or processed in a mannercomparable to baking, i.e., to dry or harden by subjecting to heat.Examples of a baked good product include, but are not limited to: bread,cakes, doughnuts, bars, pastas, bread crumbs, baked snacks,mini-biscuits, mini-crackers, mini-cookies, and mini-pretzels. As wasmentioned above, oils from the recombinant EPA production host cells canbe used as an ingredient.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

The beverage can be in a liquid or in a dry powdered form. For example,there can be mentioned: non-carbonated drinks; fruit juices, fresh,frozen, canned or concentrate; flavored or plain milk drinks, etc. Adultand infant nutritional formulas are well known in the art andcommercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum®from Ross Products Division, Abbott Laboratories).

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to: whole milk, skim milk, fermented milk products such asyoghurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

Additional food products into which the Yarrowia biomass, partiallypurified biomass, purified oil, and/or purified EPA could be includedare, for example: chewing gums, confections and frostings, gelatins andpuddings, hard and soft candies, jams and jellies, white granulatedsugar, sugar substitutes, sweet sauces, toppings and syrups, anddry-blended powder mixes.

Infant Formulas: Infant formulas are liquids or reconstituted powdersfed to infants and young children. “Infant formula” is defined herein asan enteral nutritional product which can be substituted for human breastmilk in feeding infants and typically is composed of a desiredpercentage of fat mixed with desired percentages of carbohydrates andproteins in an aqueous solution (e.g., see U.S. Pat. No. 4,670,285).Based on worldwide composition studies, as well as levels specified byexpert groups, average human breast milk typically contains about 0.20%to 0.40% of total fatty acids (assuming about 50% of calories from fat);and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2(see, e.g., formulations of Enfamil LIPIL™ [Mead Johnson & Company] andSimilac Advance™ [Ross Products Division, Abbott Laboratories]). Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants. Althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive.

Health Food Products And Pharmaceuticals: The present biomass, partiallypurified biomass, purified oil, and/or purified EPA may be used informulations to impart health benefit in health food products, includingfunctional foods, medical foods, medical nutritionals and dietarysupplements. Additionally, Yarrowia biomass, partially purified biomass,purified oil, and/or purified EPA may be used in standard pharmaceuticalcompositions. The present engineered strains of Yarrowia lipolytica orthe microbial oils produced therefrom comprising EPA could readily beincorporated into the any of the above mentioned food products, tothereby produce e.g., a functional or medical food. For example, moreconcentrated formulations comprising EPA include capsules, powders,tablets, softgels, gelcaps, liquid concentrates and emulsions which canbe used as a dietary supplement in humans or animals other than humans.

Animal Feed Products: Animal feeds are generically defined herein asproducts intended for use as feed or for mixing in feed for animalsother than humans. The Yarrowia biomass, partially purified biomass,purified oil, and/or purified EPA described herein can be used as aningredient in various animal feeds.

More specifically, although not to be construed as limiting, it isexpected that the EPA products from the recombinant Yarrowia host cellscan be used within pet food products, ruminant and poultry food productsand aquacultural food products. Pet food products are those productsintended to be fed to a pet, such as a dog, cat, bird, reptile, rodent.These products can include the cereal and health food products above, aswell as meat and meat byproducts, soy protein products, grass and hayproducts (such as alfalfa, timothy, oat or brome grass) and vegetables.Ruminant and poultry food products are those wherein the product isintended to be fed to e.g., turkeys, chickens, cattle and swine. As withthe pet foods above, these products can include cereal and health foodproducts, soy protein products, meat and meat byproducts, and grass andhay products as listed above. Aquacultural food products (or“aquafeeds”) are those products intended to be used in aquafarming,which concerns the propagation, cultivation or farming of aquaticorganisms and/or animals in fresh or marine waters.

It is contemplated that the present engineered strains of Yarrowialipolytica that are producing high concentrations of EPA will beespecially useful to include in most animal feed formulations. Inaddition to providing necessary ω-3 PUFAs, the yeast itself is a usefulsource of protein and other nutrients (e.g., vitamins, minerals, nucleicacids, complex carbohydrates, etc.) that can contribute to overallanimal health and nutrition, as well as increase a formulation'spalatablility. Accordingly it is contemplated that the addition of yeastbiomass comprising the recombinant Yarrowia production hosts will be anexcellent additional source of feed nutrients in animal feedformulations (see U.S. Pat. Appl. Pub. No. 2009-0093543-A1 foradditional details).

It is clear then that the present engineered strains of Yarrowialipolytica that are producing high concentrations of EPA will beespecially useful to include in most aquaculture feeds. In addition toproviding necessary ω-3 and/or ω-6 PUFAs, the yeast itself is a usefulsource of protein that can increase the formulation's palatablility. Inalternate embodiments, the oils produced by the present strains of Y.lipolytica could be introduced directly into the aquaculture feedformulations, following extraction and purification from the cell mass.

There is increasing awareness that EPA is an important ω-3 fatty acid inand of itself. As a result, it is expected herein that the EPA-enrichedoils of the recombinant Yarrowia production hosts described herein willhave very broad utility in a variety of therapeutic applications, e.g.,inflammation, cardiovascular diseases, nutrient regulation of geneexpression and dyslipidemia, and specifically in the treatment ofclinical conditions including: coronary heart disease, high bloodpressure, inflammatory disorders, Type II diabetes, ulcerative colitis,Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis,depression, and attention deficit/hyperactivity disorder.

U.S. Pat. Appl. Pub. No. 2009-0093543-A1 describes additional clinicalhuman studies, relating to EPA and inflammation, EPA and cardiovasculardiseases, ω-3 PUFAs and nutrient regulation of gene expression, and ω-3PUFAs and dyslipidemia and should be referred to therein. More recently,a randomized, double-blind placebo-controlled study was performed in 110normal healthy subject, wherein subjects were provided one of thefollowing for 6 weeks, as a means to evaluate the effects of the oils oncardiovascular disease risk factors, adverse events and safetyparameters: 600 mg/day EPA, 1800 mg/day EPA, 600 mg/day DHA or olive oil(placebo) (Gillies, P., “The New Science of Omega-3 Fatty Acids:Differential Nutritional Pharmacology” Texas Human Nutrition Conference,Texas A&M University, February 2010; U.S. Provisional PatentApplications No, 61/292,915 [filed Jan. 7, 2010] and No 61/295,347 filedJan. 15, 2010], having E. I duPont de Nemours and Company AttorneyDocket Numbers CL4938USPRV and CL4938USPRV1, respectively). The 600 mgEPA per day supplement was found to maintain healthy cholesterol levelsalready in the normal range. Notably, the EPA oils of the study werederived from engineered strains of Yarrowa lipolytica.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and, 3) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience, Hoboken, N.J. (1987).

Materials and Methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2nd ed., Sinauer Associates: Sunderland, Mass.(1989). All reagents, restriction enzymes and materials used for thegrowth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), NewEngland Biolabs, Inc. (Beverly, Mass.), GIBCO/BRL (Gaithersburg, Md.),or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.E. coli strains were typically grown at 37° C. on Luria Bertani [“LB”]plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). When PCR or site-directed mutagenesis wasinvolved in subcloning, the constructs were sequenced to confirm that noerrors had been introduced to the sequence. PCR products were clonedinto Promega's pGEM-T-easy vector (Madison, Wis.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s), “kB” means kilobase(s), “DCW” means dry cell weight, and “TFAs”means total fatty acids.

Nomenclature for Expression Cassettes

The structure of an expression cassette will be represented by a simplenotation system of “X::Y::Z”, wherein X describes the promoter fragment,Y describes the gene fragment, and Z describes the terminator fragment,which are all operably linked to one another.

Transformation and Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strain ATCC #20362 was purchased from the AmericanType Culture Collection (Rockville, Md.). Yarrowia lipolytica strainswere routinely grown at 28-30° C. in several media, according to therecipes shown below. Agar plates were prepared as required by additionof 20 g/L agar to each liquid media, according to standard methodology.

-   YPD agar medium (per liter): 10 g of yeast extract [Difco], 20 g of    Bacto peptone [Difco], and 20 g of glucose.-   Basic Minimal Media [“MM”] (per liter): 20 g glucose, 1.7 g yeast    nitrogen base without amino acids, 1.0 g proline, and pH 6.1 (do not    need to adjust).-   Minimal Media+Uracil [“MM+uracil or MMU”] (per liter): Prepare MM    media as above and add 0.1 g uracil and 0.1 g uridine.-   Minimal Media+Uracil+Sulfonylurea [“MMU+SU”] (per liter): Prepare    MMU media as above and add 280 mg sulfonylurea.-   Minimal Media+Uracil+Lysine [“MMUraLys”] (per liter): Prepare MM    media as above and add 0.1 g uracil, 0.1 g uridine. and 0.1 g    lysine.-   Minimal Media+5-Fluoroorotic Acid [“MM+5-FOA”] (per liter): 20 g    glucose, 6.7 g Yeast Nitrogen base, 75 mg uracil, 75 mg uridine and    appropriate amount of FOA (Zymo Research Corp., Orange, Calif.),    based on FOA activity testing against a range of concentrations from    100 mg/L to 1000 mg/L (since variation occurs within each batch    received from the supplier).-   High Glucose Media [“HGM”] (per liter): 80 glucose, 2.58 g KH₂PO₄    and 5.36 g K₂HPO₄, pH 7.5 (do not need to adjust).-   Fermentation medium without Yeast Extract [“FM without YE”] (per    liter): 6.70 g/L Yeast nitrogen base, 6.00 g KH₂PO₄, 2.00 g K₂HPO₄,    1.50 g MgSO₄.7H₂O, and 20 g glucose.-   Fermentation medium [“FM”] (per liter): 6.70 g/L Yeast nitrogen    base, 6.00 g KH₂PO₄, 2.00 g K₂HPO₄, 1.50 g MgSO₄.7H₂O, 20 g glucose    and 5.00 g Yeast extract (BBL).

Transformation of Y. lipolytica was performed as described in U.S. Pat.Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein by reference.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid [“FA”] analysis, cells were collected by centrifugationand lipids were extracted as described in Bligh, E. G. & Dyer, W. J.(Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters[“FAMEs”] were prepared by transesterification of the lipid extract withsodium methoxide (Roughan, G., and Nishida I., Arch Biochem Biophys.,276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia cells (0.5 mL culture)were harvested, washed once in distilled water, and dried under vacuumin a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) and a knownamount of C15:0 triacylglycerol (C15:0 TAG; Cat. No. T-145, Nu-CheckPrep, Elysian, Minn.) was added to the sample, and then the sample wasvortexed and rocked for 30 min at 50° C. After adding 3 drops of 1 MNaCl and 400 μl hexane, the sample was vortexed and spun. The upperlayer was removed and analyzed by GC.

FAME peaks recorded via GC analysis were identified by their retentiontimes, when compared to that of known fatty acids, and quantitated bycomparing the FAME peak areas with that of the internal standard (C15:0TAG) of known amount. Thus, the approximate amount (μg) of any fattyacid FAME [“μg FAME”] is calculated according to the formula: (area ofthe FAME peak for the specified fatty acid/area of the standard FAMEpeak)*(μg of the standard C15:0 TAG), while the amount (μg) of any fattyacid [“μg FA”] is calculated according to the formula: (area of the FAMEpeak for the specified fatty acid/area of the standard FAME peak)*(μg ofthe standard C15:0 TAG)*0.9503, since 1 μg of C15:0 TAG is equal to0.9503 μg fatty acids. Note that the 0.9503 conversion factor is anapproximation of the value determined for most fatty acids, which rangebetween 0.95 and 0.96.

The lipid profile, summarizing the amount of each individual fatty acidas a weight percent of TFAs, was determined by dividing the individualFAME peak area by the sum of all FAME peak areas and multiplying by 100.

Analysis of Total Lipid Content and Composition in Yarrowia lipolyticaby Flask Assay

For a detailed analysis of the total lipid content and composition in aparticular strain of Y. lipolytica, flask assays were conducted asfollowed. Specifically, one loop of freshly streaked cells wasinoculated into 3 mL FM medium and grown overnight at 250 rpm and 30° C.The OD_(600nm) was measured and an aliquot of the cells were added to afinal OD_(600nm) of 0.3 in 25 mL FM medium in a 125 mL flask. After 2days in a shaker incubator at 250 rpm and at 30° C., 6 mL of the culturewas harvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for fatty acid analysis (supra) and 10 mL dried fordry cell weight [“DCW”] determination.

For DCW determination, 10 mL culture was harvested by centrifugation for5 min at 4000 rpm in a Beckman GH-3.8 rotor in a Beckman GS-6Rcentrifuge. The pellet was resuspended in 25 mL of water andre-harvested as above. The washed pellet was re-suspended in 20 mL ofwater and transferred to a pre-weighed aluminum pan. The cell suspensionwas dried overnight in a vacuum oven at 80° C. The weight of the cellswas determined.

Total lipid content of cells [“TFAs % DCW”] is calculated and consideredin conjunction with data tabulating the concentration of each fatty acidas a weight percent of TFAs [“% TFAs”] and the EPA content as a percentof the dry cell weight [“EPA % DCW”]. Data from flask assays will bepresented as a table that summarizes the total dry cell weight of thecells [“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].More specifically, fatty acids will be identified as 16:0 (palmitate),16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2(LA), ALA, EDA, DGLA, ARA, ETrA, ETA, EPA and other.

Example 1 Generation of Yarrowia lipolytica Strain L135 (Ura3+, Leu−,Δpex3) to Produce About 46% DGLA of Total Fatty Acids

The present Example describes the construction of strain L135, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 46%DGLA relative to the total lipids via expression of a Δ9 elongase/Δ8desaturase pathway.

Briefly, as diagrammed in FIG. 2, strain L135 was derived from Yarrowialipolytica ATCC #20362 via construction of strain Y2224 (a FOA resistantmutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowiastrain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu−phenotype), strain Y4001U1 (Leu− and Ura−), strain Y4036 (producing 18%DGLA with a Leu− phenotype) and strain Y4036U (Leu− and Ura−). Furtherdetails regarding the construction of strains Y2224, Y4001, Y4001U,Y4036 and Y4036U are described in the General Methods of U.S. Pat. App.Pub. No. 2008-0254191, hereby incorporated herein by reference.

The final genotype of strain Y4036U with respect to wild type Yarrowialipolytica ATCC #20362 was Ura3−, YAT1::ME3S::Pex16, EXP1::EgD9eS::Lip1,FBAINm::EgD9eS::Lip2, GPAT::EgD9e::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, GPD::FmD12::Pex20, YAT1::FmD12::OCT (wherein FmD12is a Fusarium moniliforme Δ12 desaturase gene [U.S. Pat. No. 7,504,259];ME3S is a codon-optimized C_(16/18) elongase gene, derived fromMortierella alpina [U.S. Pat. No. 7,470,532]; EgD9e is a Euglenagracilis Δ9 elongase gene [U.S. Pat. No. 7,645,604]; EgD9eS is acodon-optimized Δ9 elongase gene, derived from Euglena gracilis [U.S.Pat. No. 7,645,604]; EgD8M is a synthetic mutant Δ8 desaturase [U.S.Pat. No. 7,709,239], derived from Euglena gracilis [U.S. Pat. No.7,256,033]).

Generation of L135 Strain with Chromosomal Deletion of Pex3

Construction of strain L135 is described in Example 12 of Intl. App.Pub. No. WO 2009/046248, hereby incorporated herein by reference.Briefly, construct pY157 was used to knock out the chromosomal geneencoding the peroxisome biogenesis factor 3 protein [peroxisomalassembly protein Peroxin 3 or “Pex3p”] in strain Y4036U, therebyproducing strain L135 (also referred to as strain Y4036 (Δpex3)).Knockout of the chromosomal Pex3 gene in strain L135, as compared to instrain Y4036 (whose native Pex3p had not been knocked out) resulted inthe following: higher lipid content (TFAs % DCW) (ca. 6.0% versus 4.7%),higher DGLA % TFAs (46% versus 19%), higher DGLA % DCW (ca. 2.8% versus0.9%) and reduced LA % TFAs (12% versus 30%). Additionally, the Δ9elongase percent conversion efficiency was increased from ca. 48% instrain Y4036 to 83% in strain L135.

The final genotype of strain L135 with respect to wildtype Yarrowialipolytica ATCC #20362 was Ura3+, Leu−, Pex3−, unknown1-,YAT1::ME3S::Pex16, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,GPAT::EgD9e::Lip2, FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::OCT.

Example 2 Generation of Yarrowia lipolytica Strains Producing from About18% to About 41% ARA of Total Fatty Acids [“TFAs”]

The present Example describes the construction of strain Y8006, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 41% ARArelative to the total lipids via expression of a Δ9 elongase/Δ8desaturase pathway.

The development of strain Y8006 (FIG. 2) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135 (described inExample 1), as well as construction of strains L135U9 and Y8002.

Generation of L135U9 (Leu−, Ura3−) Strain

Strain L135U was created via temporary expression of the Cre recombinaseenzyme in plasmid pY116 (FIG. 3; SEQ ID NO:127; described in Example 7of Intl. App. Pub. No. WO 2008/073367, hereby incorporated herein byreference) within strain L135 to produce a Leu− and Ura− phenotype.Plasmid pY116 was used for transformation of freshly grown L135 cellsaccording to the General Methods. The transformant cells were platedonto MMLeuUra and maintained at 30° C. for 3 to 4 days. Three colonieswere picked, inoculated into 3 mL liquid YPD media at 30° C. and shakenat 250 rpm/min for 1 day. The cultures were diluted to 1:50,000 withliquid MMLeuUra media, and 100 μL was plated onto new YPD plates andmaintained at 30° C. for 2 days. Eight colonies were picked from each ofthree plates (24 colonies total) and streaked onto MMLeu and MMLeuUraselection plates. The colonies that could grow on MMLeuUra plates butnot on MMLeu plates were selected and analyzed by GC to confirm thepresence of C20:2 (EDA). One strain, having a Leu− and Ura− phenotype,was designated as L135U9.

Generation of Y8002 Strain to Produce About 32% ARA of TFAs

Construct pZKSL-555A5 (FIG. 4A; SEQ ID NO:128) was generated tointegrate three Δ5 desaturase genes into the Lys loci of strain L135U9,to thereby enable production of ARA. The pZKSL-555A5 plasmid containedthe following components:

TABLE 6 Description of Plasmid pZKSL-5S5A5 (SEQ ID NO: 128) RE Sites AndNucleotides Within SEQ ID NO: 128 Description Of Fragment And ChimericGene Components AscI/BsiWI 720 by 5′ portion of Yarrowia Lys5 gene(GenBank Accession (5925-6645) No. M34929; labeled as “lys5 5′ region”in Figure) PacI/SphI 689 by 3′ portion of Yarrowia Lys5 gene (GenBankAccession (2536-3225) No. M34929; labeled as “Lys5-3′” in Figure)EcoRI/BsiWI FBAIN::EgD5SM::Pex20, comprising: (9338-6645) FBAIN:Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356); EgD5SM:Synthetic mutant Δ5 desaturase (SEQ ID NO: 71; U.S. Pat. Pub. No.2010-0075386-A1), derived from Euglena gracilis (U.S. Pat. No.7,678,560) (labeled as “ED5S” in Figure); Pex20: Pex20 terminatorsequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613)PmeI/ClaI YAT1::EaD5SM::OCT, comprising: (11503-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2006-0094102-A1); EaD5SM: Synthetic mutant Δ5 desaturase (SEQID NO: 81; U.S. Pat. Pub. No. 2010-0075386-A1), derived from Euglenaanabaena (U.S. Pat. Appl. Pub. No. 2008-0274521-A1) (labeled as “EaD5S”in Figure); OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) ClaI/PacI EXP1::EgD5M::Pex16, comprising: (1-2536)EXP1: Yarrowia lipolytica export protein (EXP1) promoter (labeled as“EXP” in Figure; Intl. App. Pub. No. WO 2006/052870); EgD5M: Mutant Δ5desaturase (SEQ ID NO: 69; U.S. Pat. Pub. No. 2010-0075386-A1) withelimination of internal EcoRI, BglII, HindIII and NcoI restrictionenzyme sites, derived from Euglena gracilis (U.S. Pat. No. 7,678,560)(labeled as “Euglena D5WT” in Figure); Pex16: Pex16 terminator sequencefrom Yarrowia Pex16 gene (GenBank Accession No. U75433) EcoRI/PmeIYarrowia Leu2 gene (GenBank Accession No. M37309) (9360-11503)

The pZKSL-5S5A5 plasmid was digested with AscI/SphI, and then used fortransformation of strain L135U9 according to the General Methods. Thetransformant cells were plated onto MMUraLys plates and maintained at30° C. for 2 to 3 days. Single colonies were then re-streaked ontoMMUraLys plates, and then inoculated into liquid MMUraLys at 30° C. andshaken at 250 rpm/min for 2 days. The cells were subjected to fatty acidanalysis, according to the General Methods.

GC analyses showed the presence of ARA in the transformants containingthe 3 chimeric genes of pZKSL-555A5, but not in the parent L135U9strain. Five strains (i.e., #28, #62, #73, #84 and #95) that producedabout 32.2%, 32.9%, 34.4%, 32.1% and 38.6% ARA of TFAs were designatedas strains Y8000, Y8001, Y8002, Y8003 and Y8004, respectively. Furtheranalyses showed that the three chimeric genes of pZKSL-5S5A5 were notintegrated into the Lys5 site in the Y8000, Y8001, Y8002, Y8003 andY8004 strains. All strains possessed a Lys+ phenotype.

The final genotype of strains Y8000, Y8001, Y8002, Y8003 and Y8004 withrespect to wildtype Yarrowia lipolytica ATCC #20362 was Ura−, Pex3−,unknown 1-, unknown 2-, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct.

Generation of Y8006 Strain to Produce About 41% ARA of TFAs

Construct pZP3-Pa777U (FIG. 4B; SEQ ID NO:129; described in Table 9 ofU.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein byreference) was generated to integrate three Δ17 desaturase genes intothe Pox3 loci (GenBank Accession No. AJ001301) of strain Y8002.

The pZP3-Pa777U plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8002 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 2 to 3 days. Single colonies were then re-streaked onto MM plates,and then inoculated into liquid MM at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were subjected to fatty acid analysis, accordingto the General Methods.

GC analyses showed the presence of 26% to 31% EPA of TFAs in most of theselected 96 transformants containing the 3 chimeric genes ofpZP3-Pa777U, but not in the parent Y8002 strain. Strain #69 producedabout 38% EPA of TFAs and was designated as Y8007. There was one strain(i.e., strain #9) that did not produce EPA, but produced about 41% ARAof TFAs. This strain was designated as Y8006. Based on the lack of EPAproduction in strain Y8006, its genotype with respect to wildtypeYarrowia lipolytica ATCC #20362 is assumed to be Pex3−, unknown 1-,unknown 2-, unknown 3-, Leu+, Lys+, Ura+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct.

In contrast, the final genotype of strain Y8007 with respect to wildtypeYarrowia lipolytica ATCC #20362 was Pex3−, unknown 1-, unknown 2-,unknown 3-, Leu+, Lys+, Ura+, YAT1::ME3S::Pex16, GPD::FmD12::Pex20,YAT1::FmD12::Oct, GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16,FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco (whereinPaD17 is a Pythium aphanidermatum Δ17 desaturase [U.S. Pat. No.7,556,949] and PaD17S is a codon-optimized Δ17 desaturase, derived fromPythium aphanidermatum [U.S. Pat. No. 7,556,949].

Integration of the 3 chimeric genes of pZP3-Pa777U into the Pox3 loci(GenBank Accession No. AJ001301) in strains Y8006 and Y8007 was notconfirmed.

Example 3 Generation of Yarrowia lipolytica Strains Producing from About24% to About 56% EPA of Total Fatty Acids [“TFAs”]

The present Example describes the construction of strain Y8412, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 56% EPArelative to the total lipids via expression of a Δ9 elongase/Δ8desaturase pathway.

The development of strain Y8412 (FIG. 2) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135 (described inExample 1), strains L135U9 and Y8002 (described in Example 2), andstrains Y8006U6, Y8069, Y8069U, Y8154, Y8154U, Y8269 and Y8269U.

Generation of Strain Y8006U6 (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1, hereby incorporated herein by reference) was used tointegrate a Ura3 mutant gene into the Ura3 gene of strain Y8006.

Plasmid pZKUM was digested with SalI/Pact, and then used to transformstrain Y8006 according to the General Methods. Following transformation,cells were plated onto MM+5-FOA selection plates and maintained at 30′Cfor 2 to 3 days.

A total of 8 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. All 8strains had a Ura− phenotype (i.e., cells could grow on MM+5-FOA plates,but not on MM plates). The cells were scraped from the MM+5-FOA platesand subjected to fatty acid analysis, according to the General Methods.

GC analyses showed the presence of 22.9%, 25.5%, 23.6% 21.6%, 21.6% and25% ARA in the pZKUM-transformant strains #1, #2, #4, #5, #6 and #7,respectively, grown on MM+5-FOA plates. These six strains weredesignated as strains Y8006U1, Y8006U2, Y8006U3, Y8006U4, Y8006U5 andY8006U6, respectively (collectively, Y8006U).

Generation of Y8069 Strain to Produce About 37.5% EPA of TFAs

Construct pZP3-Pa777U (FIG. 4B; SEQ ID NO:129; described in Table 9 ofU.S. Pat. Appl. Pub. No. 2009-0093543-A1, hereby incorporated herein byreference) was used to integrate three Δ17 desaturase genes into thePox3 loci (GenBank Accession No. AJ001301) of strain Y8006U6.

The pZP3-Pa777U plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8006U6 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 2 to 3 days. Single colonies were then re-streaked onto MM plates,and then inoculated into liquid MM at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were subjected to fatty acid analysis, accordingto the General Methods.

GC analyses showed the presence of EPA in the transformants containingthe 3 chimeric genes of pZP3-Pa777U, but not in the parent Y8006U6strain. Most of the selected 24 strains produced 24-37% EPA of TFAs.Four strains (i.e., #1, #6, #11 and #14) that produced 37.5%, 43.7%,37.9% and 37.5% EPA of TFAs were designated as Y8066, Y8067, Y8068 andY8069, respectively. Integration of the 3 chimeric genes of pZP3-Pa777Uinto the Pox3 loci (GenBank Accession No. AJ001301) of strains Y8066,Y8067, Y8068 and Y8069 was not confirmed.

The final genotype of strains Y8066, Y8067, Y8068 and Y8069 with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-,unknown 2-, unknown 3-, unknown 4-, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct, YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16,FBAINm::PaD17::Aco.

Generation of Strain Y8069U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8069, in a manner similar to that described for pZKUMtransformation of strain Y8006 (supra). A total of 3 transformants weregrown and identified to possess a Ura− phenotype.

GC analyses showed the presence of 22.4%, 21.9% and 21.5% EPA in thepZKUM-transformant strains #1, #2 and #3, respectively, grown onMM+5-FOA plates. These three strains were designated as strains Y8069U1,Y8069U2, and Y8069U3, respectively (collectively, Y8069U).

Generation of Strain Y8154 to Produce about 44.8% EPA of TFAs

Construct pZKL2-5mB89C (FIG. 5B; SEQ ID NO:131) was generated tointegrate one Δ5 desaturase gene, one Δ9 elongase gene, one Δ8desaturase gene, and one Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip2 loci (GenBankAccession No. AJ012632) of strain Y8069U3 to thereby enable higher levelproduction of EPA. The pZKL2-5mB89C plasmid contained the followingcomponents:

TABLE 7 Description of Plasmid pZKL2-5mB89C (SEQ ID NO: 131) RE SitesAnd Nucleotides Within SEQ ID NO: 131 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 722 bp 5′ portion of Yarrowia Lip2gene (labeled as “Lip2.5N” in (730-1) Figure; GenBank Accession No.AJ012632) PacI/SphI 697 bp 3′ portion of Yarrowia Lip2 gene (labeled as“Lip2.3N” in (4141-3438) Figure; GenBank Accession No. AJ012632)SwaI/BsiWI YAT1::YICPT1::Aco, comprising: (13561-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2006-0094102-A1); YICPT1: Yarrowia lipolytica diacylglycerolcholinephosphotransferase gene (SEQ ID NO: 37) (labeled as “Y.lipolytica CPT1 cDNA” in Figure; Intl. App. Pub. No. WO 2006/052870);Aco: Aco terminator sequence from Yarrowia Aco gene (GenBank AccessionNo. AJ001300) PmeI/SwaI FBAIN::EgD8M::Lip1 comprising: (10924-13561)FBAIN: Yarrowia lipolytica FBAIN promoter (U.S. Pat. No. 7,202,356);EgD8M: Synthetic mutant Δ8 desaturase (SEQ ID NO: 59; U.S. Pat. No.7,709,239), derived from Euglena gracilis (“EgD8S”; U.S. Pat. No.7,256,033) (labeled as “D8S-23” in Figure); Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020)PmeI/ClaI YAT1::EgD9eS::Lip2, comprising: (10924-9068) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2006-0094102-A1); EgD9eS: codon-optimized Δ9 elongase (SEQ IDNO: 45), derived from Euglena gracilis (U.S. Pat. No. 7,645,604); Lip2:Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) ClaI/EcoRI Yarrowia Ura3 gene (GenBank Accession No. AJ306421)(9068-6999) EcoRI/PacI GPDIN::EgD5SM::ACO, comprising: (6999-4141)GPDIN: Yarrowia lipolytica GPDIN promoter (U.S. Pat. No. 7,459,546);EgD5SM: Synthetic mutant Δ5 desaturase (SEQ ID NO: 71; U.S. Pat. Pub.No. 2010-0075386-A1), derived from Euglena gracilis (U.S. Pat. No.7,678,560) (labeled as “EgD5S-HPGS” in Figure); Aco: Aco terminatorsequence from Yarrowia Aco gene (GenBank Accession No. AJ001300)

The pZKL2-5mB89C plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8069U3 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains producedapproximately 38-44% EPA of TFAs. Seven strains (i.e., #1, #39, #49,#62, #70, #85 and #92) that produced about 44.7%, 45.2%, 45.4%, 44.8%,46.1%, 48.6% and 45.9% EPA of TFAs were designated as strains Y8151,Y8152, Y8153, Y8154, Y8155, Y8156 and Y8157, respectively. Knockout ofthe Lip2 gene was not confirmed in these EPA strains.

The final genotype of strains Y8151, Y8152, Y8153, Y8154, Y8155, Y8156and Y8157 with respect to wildtype Yarrowia lipolytica ATCC #20362 wasUra+, Pex3−, unknown 1-, unknown 2-, unknown 3-, unknown 4-, unknown 5-,Leu+, Lys+, YAT1::ME3S::Pex16, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct,FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT::Aco.

Generation of Strain Y8154U1 (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8154, in a manner similar to that described for pZKUMtransformation of strain Y8006 (supra). A total of 8 transformants weregrown and identified to possess a Ura− phenotype.

GC analyses showed that there was 23.1% EPA of TFAs in thepZKUM-transformant strain #7. This strain was designated as strainY8154U1.

Generation of Strain Y8269 to Produce About 45.3% EPA of TFAs

Construct pZKL1-2SR9G85 (FIG. 6A; SEQ ID NO:132) was generated tointegrate one DGLA synthase, one Δ12 desaturase gene and one Δ5desaturase gene into the Lip1 loci (GenBank Accession No. Z50020) ofstrain Y8154U1 to thereby enable higher level production of EPA. A DGLAsynthase is a multizyme comprising a Δ9 elongase linked to a Δ8desaturase.

The pZKL1-2SR9G85 plasmid contained the following components:

TABLE 8 Description of Plasmid pZKL1-2SR9G85 (SEQ ID NO: 132) RE SitesAnd Nucleotides Within SEQ ID NO: 132 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 809 bp 5′ portion of Yarrowia Lip1gene (labeled as “Lip1-5′N” in (4189-3373) Figure; GenBank Accession No.Z50020) PacI/SphI 763 bp 3′ portion of Yarrowia Lip1 gene (labeled as“Lip1.3N” in (7666-6879) Figure; GenBank Accession No. Z50020) ClaI/SwaIYAT1::E389D9eS/EgD8M::Lip1, comprising: (1-3217) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2006-0094102-A1); E389D9eS/EgD8M: gene fusion comprising acodon- optimized Δ9 elongase derived from Eutreptiella sp. CCMP389(“E389D9eS”), a linker, and the synthetic mutant Δ8 desaturase derivedfrom Euglena gracilis (“EgD8M”) (SEQ ID NO: 12) (labeled as individuallyas “E389S”, “Linker” and “EgD8M” in Figure; U.S. Pat. Appl. Pub. No.2008- 0254191-A1); Lip1: Lip1 terminator sequence from Yarrowia Lip1gene (GenBank Accession No. Z50020) SalI/ClaI GPM::EgD5SM::Octcomprising: (11982-1) GPM: Yarrowia lipolytica GPM promoter (labeled as“GPML” in Figure; U.S. Pat. No. 7,202,356); EgD5SM: Synthetic mutant Δ5desaturase (SEQ ID NO: 71; U.S. Pat. Pub. No. 2010-0075386-A1), derivedfrom Euglena gracilis (U.S. Pat. No. 7,678,560) (labeled as “ED5S” inFigure); OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) SalI/EcoRI Yarrowia Ura3 gene (GenBank AccessionNo. AJ306421) (11982-10363) EcoRI/PacI EXP1::FmD12S::ACO, comprising:(10363-7666) EXP1: Yarrowia lipolytica export protein (EXP1) promoter(labeled as “Exp” in Figure; Intl. App. Pub. No. WO 2006/052870);FmD12S: codon-optimized Δ12 elongase (SEQ ID NO: 93), derived fromFusarium moniliforme (labeled as “FD12S” in Figure; U.S. Pat. No.7,504,259); Aco: Aco terminator sequence from Yarrowia Aco gene (GenBankAccession No. AJ001300)

The pZKL1-2SR9G85 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8154U1 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains produced40-44.5% EPA of total lipids. Five strains (i.e., #44, #46, #47, #66 and#87) that produced about 44.8%, 45.3%, 47%, 44.6% and 44.7% EPA of TFAswere designated as Y8268, Y8269, Y8270, Y8271 and Y8272, respectively.Knockout of the Lip1 loci (GenBank Accession No. Z50020) was notconfirmed in these EPA strains.

The final genotype of strains Y8268, Y8269, Y8270, Y8271 and Y8272 withrespect to wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−,unknown 1-, unknown 2-, unknown 3-, unknown 4-, unknown 5-, unknown6-,YAT1::ME3S::Pex16, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, YAT1::E389D9eS/EgD8M::Lip1,GPD::FmD12::Pex20, YAT1::FmD12::Oct, EXP1::FmD12S::Aco,EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct, FBAIN::EgD5SM::Pex20,GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT::Aco.

Generation of Strain Y8269U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8269, in a manner similar to that described for pZKUMtransformation of strain Y8006 (supra). A total of 8 transformants weregrown and identified to possess a Ura− phenotype.

GC analyses showed that there were 23.0%, 23.1% and 24.2% EPA of TFAs inpZKUM-transformant strains #2, #3 and #5, respectively. These strainswere designated as strains Y8269U1, Y8269U2 and Y8269U3, respectively(collectively, Y8269U).

Generation of Strain Y8406 and Strain Y8412 To Produce About 51.2% EPAand 55.8% EPA of TFAs

Construct pZSCP-Ma83 (FIG. 6B; SEQ ID NO:133) was generated to integrateone Δ8 desaturase gene, one C_(16/18) elongase gene and one malonyl-CoAsynthetase gene into the SCP2 loci (GenBank Accession No. XM 503410) ofstrain Y8269U1 to thereby enable higher level production of EPA. ThepZSCP-Ma83 plasmid contained the following components:

TABLE 9 Description of Plasmid pZSCP-Ma83 (SEQ ID NO: 133) RE Sites AndNucleotides Within SEQ ID NO: 133 Description Of Fragment And ChimericGene Components BsiWI/AscI 1327 bp 3′ portion of Yarrowia SCP2 gene(labeled as “SCP2-3′” (1-1328) in Figure; GenBank Accession No.XM_503410) SphI/PacI 1780 bp 5′ portion of Yarrowia SCP2 gene (labeledas “SCP2-5′” (4036-5816) in Figure; GenBank Accession No. XM_503410)SwaI/BsiWI GPD::ME3S::Pex20, comprising: (12994-1) GPD: Yarrowialipolytica GPD promoter (U.S. Pat. No. 7,259,255); ME3S: codon-optimizedC_(16/18) elongase gene (SEQ ID NO: 97), derived from M. alpina (U.S.Pat. No. 7,470,532); Pex20: Pex20 terminator sequence from YarrowiaPex20 gene (GenBank Accession No. AF054613) PmeI/SwaI YAT1::MCS::Lip1comprising: (10409-12994) YAT1: Yarrowia lipolytica YAT1 promoter(labeled as “YAT” in Figure; U.S. Pat. Appl. Pub. No. 2006/0094102-A1);MCS: codon-optimized malonyl-CoA synthetase gene (SEQ ID NO: 41),derived from Rhizobium leguminosarum bv. viciae 3841 (U.S. PatentApplication No. 12/637877); Lip1: Lip1 terminator sequence from YarrowiaLip1 gene (GenBank Accession No. Z50020) ClaI/PmeI GPD::EaD8S::Pex16comprising: (7917-10409) GPD: Yarrowia lipolytica GPD promoter (U.S.Pat. No. 7,259,255); EaD8S: codon-optimized Δ8 desaturase gene (SEQ IDNO: 63), derived from Euglena anabaena (U.S. Pat. Appl. Pub. No.2008-0254521-A1); Pex16: Pex16 terminator sequence from Yarrowia Pex16gene (GenBank Accession No. U75433) SalI/EcoRI Yarrowia Ura3 gene(GenBank Accession No. AJ306421) (7467-5848)

The pZSCP-Ma83 plasmid was digested with AscI/SphI, and then used fortransformation of strains Y8269U1, Y8269U2 and Y8269U3, separately,according to the General Methods. The transformant cells were platedonto MM plates and maintained at 30° C. for 3 to 4 days. Single colonieswere re-streaked onto MM plates, and then inoculated into liquid MM at30° C. and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, resuspended in HGM and then shaken at 250 rpm/min for 5days. The cells were subjected to fatty acid analysis, according to theGeneral Methods.

A total of 96 strains resulting from each pZSCP-Ma83 transformation(i.e., into Y8269U1, Y8269U2 and Y8269U3) were analyzed by GC. Most ofthe selected 288 strains produced 43-47% EPA of TFAs. Seven strains ofY8269U1 transformed with pZSCP-Ma83 (i.e., #59, #61, #65, #67, #70, #81and #94) that produced about 51.3%, 47.9%, 50.8%, 48%, 47.8%, 47.8% and47.8% EPA of TFAs were designated as strains Y8404, Y8405, Y8406, Y8407,Y8408, Y8409 and Y8410, respectively. Three strains of Y8269U2transformed with pZSCP-Ma83 (i.e., #4, #13 and #17) that produced about48.8%, 50.8%, and 49.3% EPA of TFAs were designated as Y8411, Y8412 andY8413, respectively.

And, two strains of Y8269U3 transformed with pZSCP-Ma83 (i.e., #2 and#16) that produced about 49.3% and 53.5% EPA of TFAs were designated asY8414 and Y8415, respectively.

Knockout of the SCP2 loci (GenBank Accession No. XM_(—)503410) instrains Y8404, Y8405, Y8406, Y8407, Y8408, Y8409, Y8410, Y8411, Y8412,Y8413, Y8414 and Y8415 was not confirmed in any of these EPA strains,produced by transformation with pZSCP-Ma83.

The final genotype of strains Y8404, Y8405, Y8406, Y8407, Y8408, Y8409,Y8410, Y8411, Y8412, Y8413, Y8414 and Y8415 with respect to wildtypeYarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-, unknown 2-,unknown 3-, unknown 4-, unknown 5-, unknown6-, unknown 7-,YAT1::ME3S::Pex16, GPD::ME3S::Pex20, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2,FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1,GPD::EaD8S::Pex16, YAT1::E389D9eS/EgD8M::Lip1, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::FmD12S::Aco, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct, FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco,GPM::EgD5SM::Oct, FBAINm::PaD17::Aco, EXP1::PaD17::Pex16,YAT1::PaD17S::Lip1, YAT1::YICPT::Aco, YAT1::MCS::Lip1.

Yarrowia lipolytica strain Y8406 was deposited with the American TypeCulture Collection on May 14, 2009 and bears the designation ATCCPTA-10025. Yarrowia lipolytica strain Y8412 was deposited with theAmerican Type Culture Collection on May 14, 2009 and bears thedesignation ATCC PTA-10026.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8404, Y8405, Y8406, Y8407, Y8408,Y8409, Y8410, Y8411, Y8412, Y8413, Y8414 and Y8415 were grown andanalyzed for total lipid content and composition, according to theGeneral Methods. Table 10 summarizes the total dry cell weight of thecells [“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].More specifically, fatty acids are identified as 16:0 (palmitate), 16:1(palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA),ALA, EDA, DGLA, ARA, ETrA, ETA, EPA and other.

TABLE 10 Total Lipid Content And Composition In Yarrowia Strains Y8404,Y8405, Y8406, Y8407, Y8408, Y8409, Y8410, Y8411, Y8412, Y8413, Y8414 AndY8415 By Flask Assay DCW TFAs % % TFAs EPA % Strain (g/L) DCW 16:0 16:118:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA other DCW Y8404 4.1 27.32.8 0.8 1.8 5.1 20.4 2.1 2.9 2.5 0.6 0.8 2.4 51.1 6.3 14.0 Y8405 3.929.6 2.7 0.5 2.9 5.7 20.5 2.8 2.7 2.1 0.5 0.7 2.0 51.4 5.1 15.2 Y84064.0 30.7 2.6 0.5 2.9 5.7 20.3 2.8 2.8 2.1 0.5 0.8 2.1 51.2 5.4 15.7Y8407 4.0 29.4 2.6 0.5 3.0 5.6 20.5 2.8 2.7 2.1 0.4 0.7 2.1 51.5 5.115.2 Y8408 4.1 29.8 2.9 0.6 2.7 5.7 20.2 2.8 2.6 2.1 0.5 0.9 2.1 51.25.5 15.3 Y8409 3.9 30.8 2.8 0.5 2.9 5.7 20.6 2.7 2.7 2.1 0.5 0.8 2.151.0 5.2 15.7 Y8410 4.0 31.8 2.7 0.5 3.0 5.7 20.5 2.9 2.7 2.1 0.5 0.72.1 50.9 5.3 16.2 Y8411 3.6 30.5 2.7 0.3 3.3 5.1 19.9 2.6 2.4 2.0 0.50.6 1.8 52.9 5.7 16.1 Y8412 3.2 27.0 2.5 0.4 2.6 4.3 19.0 2.4 2.2 2.00.5 0.6 1.9 55.8 5.6 15.1 Y8413 2.9 27.2 3.1 0.4 2.6 5.4 19.9 2.2 2.82.0 0.5 0.7 1.8 52.4 5.9 14.2 Y8414 3.7 27.1 2.5 0.7 2.3 6.0 19.9 1.63.4 3.4 0.6 0.6 3.1 49.4 6.1 13.4 Y8415 3.6 25.9 1.4 0.3 1.9 4.5 16.01.3 2.7 2.9 0.5 0.6 2.5 59.0 6.1 15.3

Example 4 Generation of Yarrowia lipolytica Strain Y8647 to ProduceAbout 53.6% EPA of Total Fatty Acids [“TFAs”] with 37.6% Total LipidContent

The present Example describes the construction of strain Y8647, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 53.6%EPA relative to the total lipids with 37.6% total lipid content [“TFAs %DCW”] via expression of a Δ9 elongase/Δ8 desaturase pathway. Thedevelopment of strain Y8647 (FIG. 2) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135 (described inExample 1), strains L135U9 and Y8002 (described in Example 2), strainsY8006U6, Y8069, Y8069U, Y8154, Y8154U, Y8269 and Y8269U (described inExample 3) and strain Y8412U6.

Generation of Strain Y8412U6 (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8412 (Example 3) in a manner similar to that describedfor pZKUM transformation of strain Y8006 (Example 3). A total of 8transformants were grown and identified to possess a Ura− phenotype.

GC analyses showed that there were 25.9% and 26.9% EPA of TFAs inpZKUM-transformant strains #4 and #6, respectively. These two strainswere designated as strains Y8412U6 and Y8412U8, respectively(collectively, Y8412U).

Generation of Strain Y8647

Construct pZKL4-398F2 (FIG. 7A; SEQ ID NO:134) was generated tointegrate one C_(16/18) elongase gene, one DGLA synthase, and one Δ12desaturase gene into the Yarrowia lipase-like locus (designated as Lip4,GenBank Accession No. XM 503825) of strain Y8412U6 to thereby enablehigher level production of EPA. The pZKL4-398F2 plasmid contained thefollowing components:

TABLE 11 Description of Plasmid pZKL4-398F2 (SEQ ID NO: 134) RE SitesAnd Nucleotides Within SEQ ID NO: 134 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 745 by 5′ portion of Yarrowia Lipase4 locus (labeled as “Lip4” in (11164-10412) Figure; GenBank AccessionNo. XM_503825) PacI/SphI 782 by 3′ portion of Yarrowia Lipase 4 locus(labeled as “Lip4-3′” (1-13872) in Figure; GenBank Accession No.XM_503825) EcoRI/PacI GPDIN::FmD12::Pex16, comprising: (2877-1) GPDIN:Yarrowia lipolytica GPDIN promoter (U.S. Pat. No. 7,459,546); FmD12:Fusarium moniliforme Δ12 desaturase (SEQ ID NO: 91) (labeled as “F.D12”in Figure; U.S. Pat. No. 7,504,259); Pex16: Pex16 terminator sequencefrom Yarrowia Pex16 gene (Gen Bank Accession No. U75433) PmeI/SwaIYAT1::ME3S::Lip1 comprising: (8361-10256) YAT1: Yarrowia lipolytica YAT1promoter (labeled as “YAT” in Figure; U.S. Pat. Appl. Pub. No.2006-0094102-A1); ME3S: codon-optimized C_(16/18) elongase gene (SEQ IDNO: 97), derived from M. alpina (U.S. Pat. No. 7,470,532); Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) SwaI/ClaI FBAINm::EaD9eS/EaD8S::Lip2 comprising: (8325-4946)FBAINm: Yarrowia lipolytica FBAINm promoter (U.S. Pat. No. 7,202,356);EaD9eS/EaD8S: gene fusion comprising a codon-optimized Δ9 elongasederived from Euglena anabaena (“EaD9eS”), a linker, and acodon-optimized Δ8 desaturase derived from Euglena anabaena (“EaD8S”)(SEQ ID NO: 63) (labeled as individually as “EaD9E9S”, “Linker” and“EaD8S” in Figure; U.S. Pat. Appl. Pub. No. 2008-0254191-A1); Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) ClaI/EcoRI Yarrowia Ura3 gene (GenBank Accession No. AJ306421)(49146-2877)

The pZKL4-398F2 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8412U6, according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 transformant strainsproduced 50-52.7% EPA of TFAs. Seven strains (i.e., #31, #35, #38, #41,#60, #61 and #95) that produced about 52.8%, 53.1%, 52.8%, 53.2%, 53.1%,52.8%, and 52.9% EPA of TFAs were designated as Y8646, Y8647, Y8648,Y8649, Y8650, Y8651 and Y8652, respectively.

Knockout of the Lip4 locus (GenBank Accession No. XM_(—)503825) in theseEPA strains was not confirmed.

The final genotype of strains Y8646, Y8647, Y8648, Y8649, Y8650, Y8651and Y8652 with respect to wildtype Yarrowia lipolytica ATCC #20362 wasUra+, Pex3−, unknown 1-, unknown 2-, unknown 3-, unknown 4-, unknown 5-,unknown6-, unknown 7-, unknown 8-, YAT1::ME3S::Pex16, GPD::ME3S::Pex20,YAT1::ME3S::Lip1, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::EaD8S::Pex16,YAT1::E389D9eS/EgD8M::Lip1, FBAINm::EaD9eS/EaD8S::Lip2,GPD::FmD12::Pex20, YAT1::FmD12::Oct, EXP1::FmD12S::Aco,GPDIN::FmD12::Pex16, EXP1::EgD5M::Pex16, FBAIN::EgD5SM:Pex20,GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct, YAT1::EaD5SM::Oct,FBAINm::PaD17::Aco, EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1,YAT1::YICPT::Aco, YAT1::MCS::Lip1.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8647, Y8648, Y8649 and Y8650 weregrown and analyzed for total lipid content and composition, according tothe General Methods. Table 12 summarizes the total dry cell weight ofthe cells [“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA,ETrA, ETA, EPA and other.

TABLE 12 Total Lipid Content And Composition In Yarrowia Strains Y8647,Y8648, Y8649 And Y8650 By Flask Assay DCW TFAs % % TFAs EPA % Strain(g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA otherDCW Y8647 3.8 37.6 1.3 0.2 2.1 4.7 20.3 1.7 3.3 3.6 0.7 0.6 3.0 53.6 4.520.1 Y8648 3.5 27.8 2.3 0.3 2.7 4.3 18.6 2.3 2.1 2.2 0.6 0.6 1.9 56.74.9 15.7 Y8649 3.6 27.9 2.4 0.3 2.9 3.7 18.8 2.2 2.1 2.4 0.6 0.8 2.155.8 5.5 15.6 Y8650 3.5 28.2 2.2 0.3 2.9 3.8 18.8 2.4 2.1 2.4 0.6 0.62.1 56.1 5.3 15.8

Example 5 Generation of Yarrowia lipolytica Strain Y9028 to ProduceAbout 54.5% EPA of Total Fatty Acids [“TFAs”] with 39.6% Total LipidContent

The present Example describes the construction of strain Y9028, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 54.5%EPA relative to the total lipids with 39.6% total lipid content [“TFAs %DCW”] via expression of a Δ9 elongase/Δ8 desaturase pathway. Thedevelopment of strain Y9028 (FIG. 2) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135 (described inExample 1), strains L135U9 and Y8002 (described in Example 2), strainsY8006U6, Y8069, Y8069U, Y8154, Y8154U, Y8269 and Y8269U (described inExample 3), strains Y8412U6 and Y8647 (described in Example 4) andstrain Y8467U.

Generation of Strain Y8647U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8647 (Example 4) in a manner similar to that describedfor pZKUM transformation of strain Y8006 (Example 3). A total of 12transformants were grown and identified to possess a Ura− phenotype.

GC analyses showed that there were 30.2%, 29.2%, 28.1% and 29.9% EPA ofTFAs in pZKUM-transformant strains #1, #3, #4 and #12, respectively.These four strains were designated as strains Y8647U1, Y8647U2, Y8647U3,and Y8647U6, respectively (collectively, Y8647U).

Generation Of Strain Y9028

Construct pZP2-85 m98F (FIG. 7B; SEQ ID NO:135) was generated tointegrate one Δ8 desaturase gene, one DGLA synthase and one Δ5desaturase gene into the Yarrowia Pox2 locus (GenBank Accession No.AJ001300) of strain Y8647U3 to thereby enable higher level production ofEPA. The pZP2-85 m98F plasmid contained the following components:

TABLE 13 Description of Plasmid pZP2-85m98F (SEQ ID NO: 135) RE SitesAnd Nucleotides Within SEQ ID NO: 135 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 810 by 5′ portion of Yarrowia Pox2gene (GenBank Accession (5986-5176) No. AJ001300) PacI/SphI 655 by 3′portion of Yarrowia Pox2 gene (GenBank Accession (9349-8694) No.AJ001300) PmeI/SwaI EXP1::EgD5SM::Lip1, comprising: (2493-5020) EXP1:Yarrowia lipolytica export protein (EXP1) promoter (labeled as “EXP” inFigure; Intl. App. Pub. No. WO 2006/052870); EgD5SM: Synthetic mutant Δ5desaturase (SEQ ID NO: 71; U.S. Pat. Pub. No. 2010-0075386-A1), derivedfrom Euglena gracilis (U.S. Pat. No. 7,678,560); Lip1: Lip1 terminatorsequence from Yarrowia Lip1 gene (GenBank Accession No. Z50020)ClaI/PmeI GPD::EaD8S::Pex16, comprising: (1-2493) GPD: Yarrowialipolytica GPD promoter (U.S. Pat. No. 7,259,255); EaD8S:codon-optimized Δ8 desaturase gene (SEQ ID NO: 63), derived from Euglenaanabaena (U.S. Pat. Appl. Pub. No. 2008-0254521-A1); Pex16: Pex16terminator sequence from Yarrowia Pex16 gene (GenBank Accession No.U75433) SalI/EcoRI Yarrowia Ura3 gene (GenBank Accession No. AJ306421)(14170-12551) EcoRI/PacI YAT1::EgD9eS/EgD8M::Aco, comprising:(12551-9349) YAT1: Yarrowia lipolytica YAT1 promoter (labeled as “YAT”in Figure; U.S. Pat. Appl. Pub. No. 2006/0094102-A1); EgD9eS/EgD8M: genefusion comprising a codon-optimized Δ9 elongase derived from Euglenagracilis (“EgD9eS”), a linker, and the synthetic mutant Δ8 desaturasederived from Euglena gracilis (“EgD8M”) (SEQ ID NO: 8) (labeled asindividually as “EgD9eS”, “Linker” and “EgD8M” in Figure; U.S. Pat.Appl. Pub. No. 2008-0254191-A1); Aco: Aco terminator sequence fromYarrowia Aco gene (GenBank Accession No. AJ001300)

The pZP2-85 m98F plasmid was digested with AscI/SphI, and then used fortransformation of strains of Y8647U1, Y8647U2, Y8647U3 and Y8647U6,individually, according to the General Methods. The transformant cellswere plated onto MM plates and maintained at 30° C. for 3 to 4 days.Single colonies were re-streaked onto MM plates, and then inoculatedinto liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cellswere collected by centrifugation, resuspended in HGM and then shaken at250 rpm/min for 5 days. The cells were subjected to fatty acid analysis,according to the General Methods.

GC analyses showed that most of the selected 48 strains of Y8647U1transformed with pZP2-85 m98F produced 49-52% EPA of TFAs. Two strains(i.e., #30 and #31) that produced about 52.6% and 52.1% EPA of TFAs weredesignated as Y9024 and Y9025, respectively.

Most of the selected 60 strains of Y8647U2 transformed with pZP2-85 m98Fproduced 49-51.9% EPA of TFAs. Strain #6 produced about 52% EPA of TFAsand was designated as Y9026.

Most of the selected 60 strains of Y8647U3 transformed with pZP2-85 m98Fproduced 50-52.2% EPA of TFAs. Six strains (i.e., #5, #6, #14, #15, #20and #34) that produced about 53.2%, 53.7%, 54.0%, 52.9%, 53.4% and 52.3%EPA of TFAs were designated as Y9027, Y9028, Y9029, Y9030, Y9031 andY9032, respectively.

Similarly, GC analyses showed that most of the selected 48 strains ofY8647U6 transformed with pZP2-85 m98F produced 50-52.1% EPA of TFAs. Twostrains (i.e., #27 and #44) that produced about 52.2% and 52.8% EPA ofTFAs were designated as Y9033 and Y9034, respectively.

Knockout of the Pox2 locus (GenBank Accession No. AJ001300) in strainsY9024, Y9025, Y9026, Y9027, Y9028, Y9029, Y9030, Y9031, Y9032, Y9033 andY9034 was not confirmed in any of these EPA strains, produced bytransformation with pZP2-85 m98F.

The final genotype of these strains with respect to wildtype Yarrowialipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-, unknown 2-, unknown3-, unknown 4-, unknown 5-, unknown6−, unknown 7-, unknown 8-,unknown9−, YAT1::ME3S::Pex16, GPD::ME3S::Pex20, YAT1::ME3S::Lip1,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, GPAT::EgD9e::Lip2,YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, EXP1::EgD8M::Pex16,FBAIN::EgD8M::Lip1, GPD::EaD8S::Pex16 (2 copies),YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAINm::EaD9eS/EaD8S::Lip2, GPD::FmD12::Pex20, YAT1::FmD12::Oct,EXP1::FmD12S::Aco, GPDIN::FmD12::Pex16, EXP1::EgD5M::Pex16,FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct,EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT::Aco,YAT1::MCS::Lip1.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y9028, Y9029 and Y9031 were grown andanalyzed for total lipid content and composition, according to theGeneral Methods.

Table 14 below summarizes the total dry cell weight of the cells[“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].More specifically, fatty acids are identified as 16:0 (palmitate), 16:1(palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA),ALA, EDA, DGLA, ARA, ETrA, ETA, EPA and other.

TABLE 14 Total Lipid Content And Composition In Yarrowia Strains Y9028,Y9029 and Y9031 By Flask Assay DCW TFAs % % TFAs EPA % Strain (g/L) DCW16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA other DCW Y90283.3 39.6 1.3 0.2 2.1 4.4 19.8 1.7 3.2 2.5 0.8 0.7 1.9 54.5 6.1 21.6Y9029 3.2 38.4 1.3 0.3 1.7 4.4 19.8 1.5 3.2 3.3 0.9 0.7 2.4 53.8 6.020.7 Y9031 3.3 38.6 1.3 0.3 1.8 4.7 20.1 1.7 3.2 3.2 0.9 0.8 2.6 52.36.3 20.2

Example 6 Generation of Yarrowia lipolytica Strains Y9481 And Y9502,Producing at Least About 57% EPA of Total Fatty Acids [“TFAs”] with atLeast About 35% Total Lipid Content

The present Example describes the construction of strains Y9481 andY9502, derived from Yarrowia lipolytica ATCC #20362 and expressing a Δ9elongase/Δ8 desaturase pathway. Strain Y9481 is capable of producingabout 60.9% EPA relative to the total lipids with 35% total lipidcontent [“TFAs % DCW”], while strain Y9502 is capable of producing about57% EPA relative to the total lipids with 37.1% TFAs % DCW.

The development of strains Y9481 and Y9502 (FIG. 2) required theconstruction of strains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135(described in Example 1), strains L135U9 and Y8002 (described in Example2), strains Y8006U6, Y8069, Y8069U, Y8154, Y8154U, Y8269 and Y8269U(described in Example 3), strains Y8412U6 and Y8647 (described inExample 4), strains Y8467U and Y9028 (described in Example 5) and strainY9028U.

Generation of Strain Y9028U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y9028 (Example 5) in a manner similar to that describedfor pZKUM transformation of strain Y8006 (Example 3). A total of 8transformants were grown and identified to possess a Ura− phenotype.

GC analyses showed that there were 24.1%, 24.9%, 24.5% and 24.5% EPA ofTFAs in pZKUM-transformant strains #1, #3, #4, and #5, respectively.These four strains were designated as strains Y9028U1, Y9028U2, Y9028U3,and Y9028U4, respectively (collectively, Y9028U).

Components of Integration Vector pZK16-ML8N

Construct pZK16-ML8N (FIG. 8A; SEQ ID NO:136) was generated to integrateone Δ8 desaturase gene, one malonyl-CoA synthetase gene, and onelysophosphatidic acid acyltransferase gene [“LPAAT”] into the YarrowiaYALI0B14795p locus (GenBank Accession No. XM_(—)500900) of strainY9028U2. The pZK16-ML8N plasmid contained the following components:

TABLE 15 Description of Plasmid pZK16-ML8N (SEQ ID NO: 136) RE Sites AndNucleotides Within SEQ ID NO: 136 Description Of Fragment And ChimericGene Components AscI/BsiWI 1904 by 5′ portion of YALI0B14795p locus(GenBank Accession (1905-1) No. XM_500900, labeled as “Y8716-5′” inFigure) PacI/SphI 1801 by 3′ portion of YALI0B14795p locus (GenBankAccession (6414-4613) No. XM_500900, labeled as “Y8716-3′” in Figure)SwaI/BsiWI YAT1::EgD8M::Pex20, comprising: (12920-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2006-0094102-A1); EgD8M: Synthetic mutant Δ8 desaturase (SEQ IDNO: 59; U.S. Pat. No. 7,709,239), derived from Euglena gracilis(“EgD8S”; U.S. Pat. No. 7,256,033); Pex20: Pex20 terminator sequencefrom Yarrowia Pex20 gene (GenBank Accession No. AF054613) PmeI/SwaIFBA::MCS::Lip1, comprising: (10534-12920) FBA: Yarrowia lipolytica FBApromoter (U.S. Pat. No. 7,202,356); MCS: codon-optimized malonyl-CoAsynthetase gene (SEQ ID NO: 41), derived from Rhizobium leguminosarumbv. viciae 3841 (U.S. Patent Application No. 12/637877); Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) ClalI/PmeI YAT1::MaLPAAT1S::Pex16, comprising: (8515-10534)YAT1: Yarrowia lipolytica YAT1 promoter (labeled as “YAT” in Figure;U.S. Pat. Appl. Pub. No. 2006-0094102-A1); MaLPAAT1S: codon-optimizedlysophosphatidic acid acyltransferase gene (SEQ ID NO: 35), derived fromMortierella alpine (U.S. Pat. Appl. Pub. No. 2006-0115881- A1; U.S. Pat.Appl. Pub. No. 2009-0325265-A1); Pex16: Pex16 terminator sequence fromYarrowia Pex16 gene (GenBank Accession No. U75433) SalI/EcoRI YarrowiaUra3 gene (GenBank Accession No. AJ306421) (8065-6446)Components of Integration Vector pZK16-ML

Construct pZK16-ML (FIG. 8B; SEQ ID NO:137) was generated to integrateone malonyl-CoA synthetase gene and one lysophosphatidic acidacyltransferase gene [“LPAAT”] into the Yarrowia YALI0B14795p locus(GenBank Accession No. XM_(—)500900) of strain Y9028U2. The componentsof the pZK16-ML plasmid are identical to those of pZK16-ML8N (supra);however, the chimeric YAT1::EgD8M::Pex20 gene of pZK16-ML8N is lacking.

Generation of Strains Y9481 and Y9502

The pZK16-ML8N plasmid and pZK16-ML plasmid were each individuallydigested with AscI/SphI, and then used separately for transformation ofstrain Y9028U2, according to the General Methods. The transformant cellswere plated onto MM plates and maintained at 30° C. for 3 to 4 days.Single colonies were re-streaked onto MM plates, and then inoculatedinto liquid MM at 30° C. and shaken at 250 rpm/min for 2 days. The cellswere collected by centrifugation, resuspended in HGM and then shaken at250 rpm/min for 5 days. The cells were subjected to fatty acid analysis,according to the General Methods.

GC analyses showed that most of the selected 96 strains of Y9028U2 withpZK16-ML8N produced 50-55.4% EPA of TFAs. Fifteen strains (i.e., #8,#18, #21, #24, #29, #48, #60, #66, #68, #75, #76, #78, #90, #95 and #96)that produced about 58.1%, 61.4%, 56.2%, 58.1%, 57.5%, 57.0%, 55.9%,57.6%, 57.8%, 55.5%, 57.6%, 58.1%, 57.1%, 56.2% and 58.6% EPA of TFAswere designated as Y9472, Y9473, Y9474, Y9475, Y9476, Y9477, Y9478,Y9479, Y9480, Y9481, Y9482, Y9483, Y9484, Y9485 and Y9486, respectively.

The final genotype of these pZK16-ML8N transformant strains with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1−,unknown 2-, unknown 3-, unknown 4-, unknown 5-, unknown6−, unknown 7-,unknown 8-, unknown9−, unknown 10-, YAT1::ME3S::Pex16, GPD::ME3S::Pex20,YAT1::ME3S::Lip1, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, YAT1::EgD8M::Pex20,GPD::EaD8S::Pex16 (2 copies), YAT1::E389D9eS/EgD8M::Lip1,YAT1::EgD9eS/EgD8M::Aco, FBAINm::EaD9eS/EaD8S::Lip2, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::FmD12S::Aco, GPDIN::FmD12::Pex16,EXP1::EgD5M::Pex16, FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco,GPM::EgD5SM::Oct, EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct,FBAINm::PaD17::Aco, EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1,YAT1::YICPT::Aco, YAT1::MCS::Lip1, FBA::MCS::Lip1,YAT1::MaLPAAT1S::Pex16.

Similarly, GC analyses showed that most of the selected 96 strains ofY9028U2 with pZK16-ML produced 51-55.5% EPA of TFAs. Sixteen strains(i.e., #4, #8, #15, #16, #39, #44, #46, #63, #66, #80, #85, #86, #88,#89, #90 and #96) that produced about 56.5%, 57.4%, 56.8%, 57.0%, 56.4%,57.3%, 58.2%, 55.6%, 57.8%, 55.6%, 57.6%, 56.8%, 55.8%, 56.4%, 56.1% and57% EPA of TFAs were designated as Y9496, Y9497, Y9498, Y9499, Y9500,Y9501, Y9502, Y9503, Y9504, Y9505, Y9506, Y9507, Y9508, Y9509, Y9510 andY9511, respectively.

The final genotype of these pZK16-ML transformant strains with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-,unknown 2-, unknown 3-, unknown 4-, unknown 5-, unknown6-, unknown 7-,unknown 8-, unknown9-, unknown 10-, YAT1::ME3S::Pex16, GPD::ME3S::Pex20,YAT1::ME3S::Lip1, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::EaD8S::Pex16 (2 copies),YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAINm::EaD9eS/EaD8S::Lip2, GPD::FmD12::Pex20, YAT1::FmD12::Oct,EXP1::FmD12S::Aco, GPDIN::FmD12::Pex16, EXP1::EgD5M::Pex16,FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct,EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT::Aco,YAT1::MCS::Lip1, FBA::MCS::Lip1, YAT1::MaLPAAT1S::Pex16.

Knockout of the YALI0B14795p locus (GenBank Accession No. XM_(—)500900)in strains Y9472, Y9473, Y9474, Y9475, Y9476, Y9477, Y9478, Y9479,Y9480, Y9481, Y94782, Y9483, Y9484, Y9485, Y9486, Y9496, Y9497, Y9498,Y9499, Y9500, Y9501, Y9502, Y9503, Y9504, Y9505, Y9506, Y9507, Y9508,Y9509, Y9510 and Y9511 was not confirmed in any of these EPA strains,produced by transformation with pZK16-ML8N or pZK16-ML.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y9477, Y9481, Y9486, Y9497, Y9502,Y9504, Y9508 and Y9510 were grown and analyzed for total lipid contentand composition, according to the General Methods.

Table 16 below summarizes the total dry cell weight of the cells[“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].More specifically, fatty acids are identified as 16:0 (palmitate), 16:1(palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (LA),ALA, EDA, DGLA, ARA, ETrA, ETA, EPA and other.

TABLE 16 Total Lipid Content And Composition In Yarrowia Strains Y9477,Y9481, Y9486, Y9497, Y9502, Y9504, Y9508 and Y9510 By Flask Assay DCWTFAs % % TFAs EPA % Strain (g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDADGLA ARA EtrA ETA EPA other DCW Y9477 3.2 32.6 2.6 0.5 3.4 4.8 10.0 0.52.5 3.7 1.0 0.5 2.1 61.4 6.9 20.0 Y9481 3.1 35.0 2.5 0.5 3.1 4.7 11.00.6 2.6 3.6 0.9 0.5 2.1 60.9 6.8 21.3 Y9486 3.1 32.2 2.1 0.7 1.8 4.211.9 0.6 2.9 4.2 1.2 0.7 2.4 60.3 6.7 19.4 Y9497 3.7 33.7 2.4 0.5 3.24.6 11.3 0.8 3.1 3.6 0.9 0.7 2.3 58.7 7.1 19.8 Y9502 3.8 37.1 2.5 0.52.9 5.0 12.7 0.9 3.5 3.3 0.8 0.7 2.4 57.0 7.5 21.3 Y9504 3.7 33.7 2.20.5 3.0 4.5 11.3 0.7 2.9 3.5 0.9 0.7 2.3 59.9 7.1 20.1 Y9508 3.7 34.92.3 0.5 2.7 4.4 13.1 0.9 2.9 3.3 0.9 0.7 2.3 58.7 7.3 20.5 Y9510 3.635.1 2.5 0.5 2.7 4.4 11.7 0.7 2.9 3.7 0.9 0.7 2.3 58.9 7.8 20.7

Example 7 Generation of Yarrowia lipolytica Strain Y8672 to ProduceAbout 61.8% EPA of Total Fatty Acids [“TFAs”] with 26.5% Total LipidContent

The present Example describes the construction of strain Y8672, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing about 61.8%EPA relative to the total lipids with 26.5% total lipid content [“TFAs %DCW”] via expression of a Δ9 elongase/Δ8 desaturase pathway. Thedevelopment of strain Y8672 (FIG. 9) required the construction ofstrains Y2224, Y4001, Y4001U, Y4036, Y4036U and L135 (described inExample 1), strains L135U9 and Y8002 (described in Example 2), strainsY8006U6, Y8069, Y8069U (described in Example 3) and strains Y8145,Y8145U, Y8259, Y8259U, Y8367 and Y8367U.

Generation of Strain Y8145 to Produce About 48.5% EPA of TFAs

Construct pZKL2-5 m89C (FIG. 10; SEQ ID NO:138) was generated tointegrate one Δ5 desaturase gene, one Δ9 elongase gene, one Δ8desaturase gene, and one Y. lipolytica diacylglycerolcholinephosphotransferase gene (CPT1) into the Lip2 loci (GenBankAccession No. AJ012632) of strain Y8069U3 (Example 3) to thereby enablehigher level production of EPA. The pZKL2-5m89C plasmid contained thefollowing components:

TABLE 17 Description of Plasmid pZKL2-5m89C (SEQ ID NO: 138) RE SitesAnd Nucleotides Within SEQ ID NO: 138 Description Of Fragment AndChimeric Gene Components AscI/BsiWI 722 by 5′ portion of Yarrowia Lip2gene (labeled as “Lip2.5N” in (730-1) Figure; GenBank Accession No.AJ012632) PacI/SphI 697 by 3′ portion of Yarrowia Lip2 gene (labeled as“Lip2.3N” in (4141-3438) Figure; GenBank Accession No. AJ012632)SwaI/BsiWI GPD::YICPT1::Aco, comprising: (13143-1) GPD: Yarrowialipolytica GPD promoter (U.S. Pat. No. 7,259,255); YICPT1: Yarrowialipolytica diacylglycerol cholinephosphotransferase gene (SEQ ID NO: 37)(Intl. App. Pub. No. WO 2006/052870); Aco: Aco terminator sequence fromYarrowia Aco gene (GenBank Accession No. AJ001300) PmeI/SwaIFBAIN::EgD8M::Lip1 comprising: (10506-13143) FBAIN: Yarrowia lipolyticaFBAIN promoter (U.S. Pat. No. 7,202,356); EgD8M: Synthetic mutant Δ8desaturase (SEQ ID NO: 59; U.S. Pat. No. 7,709,239), derived fromEuglena gracilis (“EgD8S”; U.S. Pat. No. 7,256,033); Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) PmeI/ClaI YAT1::EgD9eS::Lip2, comprising: (10506-8650) YAT1:Yarrowia lipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat.Appl. Pub. No. 2006-0094102-A1); EgD9eS: codon-optimized Δ9 elongasegene (SEQ ID NO: 45), derived from Euglena gracilis (U.S. Pat. No.7,645,604); Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene(GenBank Accession No. AJ012632) ClaI/EcoRI Yarrowia Ura3 gene (GenBankAccession No. AJ306421) (8650-6581) EcoRI/PacI YAT1::EgD5SM::ACO,comprising: (6581-4141) YAT1: Yarrowia lipolytica YAT1 promoter (labeledas “YAT” in Figure; U.S. Pat. Appl. Pub. No. 2006-0094102-A1); EgD5SM:Synthetic mutant Δ5 desaturase (SEQ ID NO: 71; U.S. Pat. Pub. No.2010-0075386-A1), derived from Euglena gracilis (U.S. Pat. No.7,678,560); Aco: Aco terminator sequence from Yarrowia Aco gene (GenBankAccession No. AJ001300)

The pZKL2-5m89C plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8069U3 according to the General Methods. Thetransformant cells were plated onto MM plates and maintained at 30° C.for 3 to 4 days. Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HGM andthen shaken at 250 rpm/min for 5 days. The cells were subjected to fattyacid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains produced38-44.5% EPA of TFAs. Four strains (i.e., #10, #50, #70 and #89) thatproduced about 45.1%, 45.6%, 45.0% and 45.6% EPA of TFAs were designatedas Y8143, Y8144, Y8145 and Y8146, respectively. Knockout of the Lip2loci (GenBank Accession No. AJ012632) was not confirmed in these EPAstrains.

The final genotype of strains Y8143, Y8144, Y8145 and Y8146 with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-,unknown 2-, unknown 3-, unknown 4-, unknown 5-, Leu+, Lys+,YAT1::ME3S::Pex16, GPD::FmD12::Pex20, YAT1::FmD12::Oct,GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1,EXP1::EgD8M::Pex16, FBAIN::EgD5SM::Pex20, YAT1::EgD5SM::Aco,EXP1::EgD5M::Pex16, YAT1::EaD5SM::Oct, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, GPD::YICPT1::Aco.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8143, Y8144, Y8145 and Y8146 weregrown and analyzed for total lipid content and composition, according tothe General Methods.

TABLE 18 Total Lipid Content And Composition In Yarrowia Strains Y8143,Y8144, Y8145 and Y8146 By Flask Assay DCW TFAs % % TFAs EPA % Strain(g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA otherDCW Y8143 4.6 22.3 4.2 1.5 1.4 3.6 18.1 2.6 1.7 1.6 0.6 2.2 1.6 50.311.6 11.2 Y8144 4.3 23 4.0 1.5 1.4 3.3 18.0 2.6 1.8 1.7 0.7 2.3 1.6 50.611.5 11.6 Y8145 4.6 23.1 4.3 1.7 1.4 4.8 18.6 2.8 2.2 1.5 0.6 2.2 1.548.5 9.9 11.2 Y8146 4.5 23.8 4.3 1.7 1.4 4.8 18.7 2.8 2.0 1.5 0.6 2.21.5 48.3 11.2 11.5

Generation of Strain Y8145U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8145 in a manner similar to that described for pZKUMtransformation of strain Y8006 (Example 3). A total of 8 transformantswere grown and identified to possess a Ura− phenotype.

GC analyses showed that there were 22.5%, 22.6% and 23.4% EPA of TFAs inpZKUM-transformant strains #5, #6 and #7, respectively. These threestrains were designated as strains Y8145U1, Y8145U2 and Y8145U3,respectively (collectively, Y8145U).

Generation of Y8259 Strain to Produce About 53.9% EPA of TFAs

Construct pZKL1-2SR9G85 (Example 3, FIG. 6A; SEQ ID NO:132) wasgenerated to integrate one DGLA synthase gene, one Δ12 desaturase geneand one Δ5 desaturase gene into the Lip1 loci (GenBank Accession No.Z50020) of strain Y8145U to thereby enable higher level production ofEPA.

The pZKL1-2SR9G85 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8145U1, in a manner similar to that describedfor pZKL1-2SR9G85 transformation of strain Y8154U1 (Example 3). Thecells were subjected to fatty acid analysis, according to the GeneralMethods.

GC analyses showed that most of the selected 96 strains produced40-44.0% EPA of total lipids. Five strains (i.e., #7, #14, #48, #56 and#60) that produced about 45.2%, 47%, 44.4%, 44.3% and 45.2% EPA of TFAswere designated as Y8255, Y8256, Y8257, Y8258 and Y8259, respectively.Knockout of the Lip1 loci (GenBank Accession No. Z50020) was notconfirmed in these EPA strains.

The final genotype of these strains with respect to wildtype Yarrowialipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-, unknown 2-, unknown3-, unknown 4-, unknown 5-, unknown 6-, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::FmD12::Pex20, YAT1::FmD12::Oct, EXP1::FmD12S::ACO,GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1,EXP1::EgD8M::Pex16, YAT1::E389S/EgD8M::Lip1, FBAIN::EgD5SM::Pex20,YAT1::EgD5SM::Aco, GPM::EgD5SM::Oct, EXP1::EgD5M::Pex16,YAT1::EaD5SM::Oct; YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16,FBAINm::PaD17::Aco, GPD::YICPT1::Aco.

Yarrowia lipolytica strain Y8259 was deposited with the American TypeCulture Collection on May 14, 2009 and bears the designation ATCCPTA-10027.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8256 and Y8259 were grown and analyzedfor total lipid content and composition, according to the GeneralMethods.

TABLE 19 Total Lipid Content And Composition In Yarrowia Strains Y8256and Y8259 By Flask Assay DCW TFAs % % TFAs EPA % Strain (g/L) DCW 16:016:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA other DCW Y8256 4.020.1 3.5 1.4 1.3 3.8 18.8 2.0 2.1 1.6 0.8 2.1 1.7 49.9 11.0 10.0 Y82594.7 20.5 3.5 1.3 1.3 4.8 16.9 2.3 1.9 1.7 0.6 1.8 1.6 53.9 8.4 11.0

Generation of Strain Y8259U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8259 in a manner similar to that described for pZKUMtransformation of strain Y8006 (Example 3). A total of 8 transformantswere grown and identified to possess a Ura− phenotype.

GC analyses showed that there was 26.6% EPA of TFAs inpZKUM-transformant strain #3. This strain was designated as strainY8259U.

Generation of Y8367 Strain to Produce About 58.3% EPA of TFAs

Construct pZP2-85 m98F (Example 5, FIG. 7B; SEQ ID NO:135) was generatedto integrate one Δ8 desaturase gene, one DGLA synthase, and one Δ5desaturase gene into the Yarrowia Pox2 locus (GenBank Accession No.AJ001300) of strain Y8259U to thereby enable higher level production ofEPA.

The pZP2-85 m98F plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8259U, in a manner similar to that describedfor pZP2-85 m98F transformation of strain Y8647U3 (Example 5). The cellswere subjected to fatty acid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains of Y8259U withpZP2-85 m98F produced 41-46% EPA of TFAs. Four strains (i.e., #26, #33,#77 and #81) that produced about 46.7%, 46.5%, 47.4% and 46.9% EPA ofTFAs were designated as Y8367, Y8368, Y8369 and Y8370, respectively.Knock out of the Pox2 locus (GenBank Accession No. AJ001300) was notconfirmed in these EPA strains.

The final genotype of strains Y8367, Y8368, Y8369 and Y8370 with respectto wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1-,unknown 2-, unknown 3-, unknown 4-, unknown 5-, unknown 6-, unknown 7-,Leu+, Lys+, YAT1::ME3S::Pex16, GPD::FmD12::Pex20, YAT1::FmD12::Oct,EXP1::FmD12S::ACO, GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,FBAIN::EgD8M::Lip1, EXP1::EgD8M::Pex16, GPD::EaD8S::Pex16,YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAIN::EgD5SM::Pex20, YAT1::EgD5SM::Aco, GPM::EgD5SM::Oct,EXP1::EgD5M::Pex16, EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco,GPD::YICPT1::Aco.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8367, Y8368, Y8369 and Y8370 weregrown and analyzed for total lipid content and composition, according tothe General Methods.

TABLE 20 Total Lipid Content And Composition In Yarrowia Strains Y8367,Y8368, Y8369 and Y8370 By Flask Assay DCW TFAs % % TFAs EPA % Strain(g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA otherDCW Y8367 3.6 18.4 3.7 1.2 1.1 3.4 14.2 1.1 1.5 1.7 0.8 2.1 1.0 58.3 9.910.7 Y8368 4.7 19.2 3.0 1.4 1.3 4.3 17.9 1.3 2.4 2.8 1.0 1.8 1.9 52.58.4 10.1 Y8369 3.5 19.7 3.7 1.2 1.6 4.2 15.6 1.8 1.7 1.9 0.6 1.7 1.655.8 8.6 11.0 Y8370 4.0 23.3 3.4 1.1 1.4 4.0 15.7 1.9 1.7 1.9 0.6 1.81.5 56.4 8.6 13.1

Generation of Strain Y8367U (Ura3−)

In order to disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ IDNO:130; described in Table 15 of U.S. Pat. Appl. Pub. No.2009-0093543-A1) was used to integrate a Ura3 mutant gene into the Ura3gene of strain Y8367 in a manner similar to that described for pZKUMtransformation of strain Y8006 (Example 3). A total of 8 transformantswere grown and identified to possess a Ura− phenotype.

GC analyses showed that there were 25.6%, 25.5% and 25.4% EPA of TFAs inpZKUM-transformant strains #2, #3 and #6, respectively. These threestrains were designated as strains Y8367U1, Y8367U2 and Y8367U3,respectively (collectively, Y8367U).

Generation of Y8672 strain to Produce About 61.8% EPA of TFAs

Construct pZSCP-Ma83 (Example 3, FIG. 6B; SEQ ID NO:133) was generatedto integrate one Δ8 desaturase gene, one C_(16/18) elongase gene and onemalonyl-CoA synthetase gene into the SCP2 loci (GenBank Accession No.XM_(—)503410) of strain Y8637U to thereby enable higher level productionof EPA.

The pZSCP-Ma83 plasmid was digested with AscI/SphI, and then used fortransformation of strain Y8367U1, in a manner similar to that describedfor pZSCP-Ma83 transformation of strain Y8269U1 (Example 3). The cellswere subjected to fatty acid analysis, according to the General Methods.

GC analyses showed that most of the selected 96 strains of Y8367U1 withpZSCP-Ma83 produced 46-52.5% EPA of TFAs. Eight strains (i.e., #8, #40,#43, #44, #61, #63, #68 and #70) that produced about 53.2%, 52.8%,52.7%, 52.9%, 53.0%, 52.6%, 53.1% and 52.7% EPA of TFAs were designatedas Y8666, Y8667, Y8668, Y8669, Y8670, Y8671, Y8672 and Y8673,respectively. Knockout of the SCP2 loci (Gen Bank Accession No.XM_(—)503410) was not confirmed in these EPA strains.

The final genotype of strains Y8666, Y8667, Y8668, Y8669, Y8670, Y8671,Y8672 and Y8673 with respect to wildtype Yarrowia lipolytica ATCC #20362was Ura+, Pex3−, unknown 1-, unknown 2-, unknown 3-, unknown 4-, unknown5-, unknown 6-, unknown 7-, unknown 8-, Leu+, Lys+, YAT1::ME3S::Pex16,GPD::ME3S::Pex20, GPD::FmD12::Pex20, YAT1::FmD12::Oct,EXP1::FmD12S::ACO, GPAT::EgD9e::Lip2, FBAINm::EgD9eS::Lip2,EXP1::EgD9eS::Lip1, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,FBAIN::EgD8M::Lip1, EXP1::EgD8M::Pex16, GPD::EaD8S::Pex16 (2 copies),YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAIN::EgD5SM::Pex20, YAT1::EgD5SM::Aco, GPM::EgD5SM::Oct,EXP1::EgD5M::Pex16, EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct,YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16, FBAINm::PaD17::Aco,GPD::YICPT1::Aco, YAT1::MCS::Lip1.

Analysis of Total Lipid Content and Composition by Flask Assay

Cells from YPD plates of strains Y8666, Y8669, Y8679 and Y8672 weregrown and analyzed for total lipid content and composition, according tothe General Methods.

TABLE 21 Total Lipid Content And Composition In Yarrowia Strains Y8666,Y8669, Y8670 And Y8672 By Flask Assay DCW TFAs % % TFAs EPA % Strain(g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPA otherDCW Y8666 3.2 25.2 2.3 0.3 2.3 4.1 15.1 1.3 1.7 1.4 0.7 0.6 1.3 62.2 6.715.6 Y8669 3.2 26.4 2.3 0.3 2.3 4.1 15.7 1.4 1.8 1.6 0.7 0.5 1.1 61.56.7 16.3 Y8670 3.2 27.3 1.9 0.4 3.4 4.3 17.0 1.5 2.2 1.7 0.6 0.5 1.160.9 4.5 16.6 Y8672 3.3 26.5 2.3 0.4 2.0 4.0 16.1 1.4 1.8 1.6 0.7 0.41.1 61.8 6.4 16.4

Example 8 Construction of Various Expression Vectors ComprisingDifferent LPLAT ORFs

The present example describes the construction of a series of vectors,each comprising a LPLAT ORF, suitable for expression in Yarrowialipolytica. LPLAT ORFs included the Saccharomyces cerevisiae Ale1,Yarrowia lipolytica Ale1, Mortierella alpina LPAAT1, Yarrowia lipolyticaLPAAT1 and Caenorhabditis elegans LPCAT. Example 9 describes the resultsobtained following transformation of these vectors into Yarrowialipolytica strain Y8406U.

Origin of LPLATs

A variety of LPLATs have been identified in the patent and openliterature, but the functionality of these genes has not been previouslydirectly compared. Table 22 summarizes publicly available LPLATs (i.e.,ScAle1, ScLPAAT, MaLPAAT1 and CeLPCAT) and LPLAT orthologs identifiedherein (i.e., YIAle1 and YILPAAT1) that are utilized in the presentExample, following codon-optimization of heterologous genes forexpression in Yarrowia lipolytica (infra).

TABLE 22 LPLATs Functionally Characterized ORF SEQ ID LPLAT OrganismDesignation References NO Ale1 Saccharomyces ORF GenBank Accession No.14, 15 cerevisiae* “YOR175C” or NP_014818; U.S. Pat. Appl. Pub. “ScAle1”No. 20080145867 (and corresponding to Intl. App. Pub. No. WO2008/076377); Intl. App. Pub. No. WO 2009/001315 Yarrowia “YALI0F19514p”GenBank Accession No. 16, 17 lipolytica or “YIAle1” XP_505624; Intl.App. Pub. No. WO 2009/001315 LPAAT Saccharomyces ORF “YDL052C” GenBankAccession No. 32 cerevisiae or “ScLPAAT” NP_010231 Mortierella“MaLPAAT1” U.S. Pat. Appl. Pub. No. 2006- 28, 29 alpina 0115881-A1; U.S.Pat. Appl. Pub. No. 2009-0325265-A1 Yarrowia “YALI0E18964g” GenBankAccession No. 30, 31 lipolytica or “YILPAAT1” XP_504127; U.S. Pat. No.7,189,559 LPCAT Caenorhabditis “clone T06E8.1” GenBank Accession No. 24,25 elegans* or “CeLPCAT” CAA98276; Intl. App. Pub. No. WO 2004/076617(corresponding to U.S. Pat. Appl. Pub. No. 2006- 0168687-A1) *TheSaccharomyces cerevisiae Ale1 and Caenorhabditis elegans LPCAT were usedas comparative Examples.

More specifically, the ScLPAAT (SEQ ID NO:32) and ScAle1 (SEQ ID NO:15)protein sequences were used as queries to identify orthologs from thepublic Y. lipolytica protein database of the “Yeast project Genolevures”(Center for Bioinformatics, LaBR1, Talence Cedex, France) (see alsoDujon, B. et al., Nature, 430(6995):35-44 (2004)). Based on analysis ofthe best hits, the Ale1 and LPAAT orthologs from Yarrowia lipolytica areidentified herein as YIAle1 (SEQ ID NO:17) and YILPAAT (SEQ ID NO:31),respectively. The identity of YIAle1 and YILPAAT1 as orthologs of ScAle1and ScLPAAT, respectively, was further confirmed by doing a reciprocalBLAST, i.e., using SEQ ID NOs:17 and 31 as a query against theSaccharomyces cerevisiae public protein database to find ScAle1 andScLPAAT, respectively, as the best hits.

The LPLAT proteins identified above as ScAle1 (SEQ ID NO:15), YIAle1(SEQ ID NO:17), ScLPAAT (SEQ ID NO:32), MaLPAAT1 (SEQ ID NO:29),YILPAAT1 (SEQ ID NO:31) and CeLPCAT (SEQ ID NO:25) were aligned usingthe method of Clustal W (slow, accurate, Gonnet option; Thompson et al.,Nucleic Acids Res., 22:4673-4680 (1994)) of the MegAlign™ program(version 8.0.2) of the LASERGENE bioinformatics computing suite(DNASTAR, Inc., Madison, Wis.). This resulted in creation of Table 23,where percent similarity is shown in the upper triangle of the Tablewhile percent divergence is shown in the lower triangle.

TABLE 23 Percent Identity And Percent Divergence Among Various LPLATsYILPAAT1 CeLPCAT MaLPAAT1 ScAle1 ScLPAAT YIAle1 —  26.6 34.0 9.6 43.911.7 YILPAAT1 184.3 — 36.4 11.3 32.4 14.5 CeLPCAT 137.5 126.4 — 11.134.6 15.0 MaLPAAT1 545.0 442.0 456.0 — 13.5 45.0 ScAle1  97.9 145.7134.5 365.0 — 15.6 ScLPAAT 426.0 339.0 330.0 94.3 317.0  — YIAle1

The percent identities revealed by this method allowed determination ofthe minimum percent identity between each of the LPAAT polypeptides andthe minimum percent identity between each of the Ale1 polypeptides. Therange of identity between LPAAT polypeptides was 34.0% identity(MaLPAAT1 and YILPAAT1) to 43.9% identity (ScLPAAT and YILPAAT1), whileidentity between the ScAle1 and YIAle1 polypeptides was 45%.

Membrane Bound O-Acyltransferase [“MBOAT”] Family Motifs: Orthologs ofthe ScAle1 protein sequence (SEQ ID NO:15) were identified by conductinga National Center for Biotechnology Information [“NCBI”]BLASTP 2.2.20(protein-protein Basic Local Alignment Search Tool; Altschul et al.,Nucleic Acids Res., 25:3389-3402 (1997); and Altschul et al., FEBS J.,272:5101-5109 (2005)) search using ScAle1 (SEQ ID NO:15) as the querysequence against fungal proteins in the “nr” protein database(comprising all non-redundant GenBank CDS translations, sequencesderived from the 3-dimensional structure from Brookhaven Protein DataBank [“PDB”], sequences included in the last major release of theSWISS-PROT protein sequence database, PIR and PRF excluding thoseenvironmental samples from WGS projects) using default parameters(expect threshold=10; word size=3; scoring parameters matrix=BLOSUM62;gap costs: existence=11, extension=1). The following hits were obtained:

TABLE 24 Fungal Orthologs Of ScAle1 (SEQ ID NO: 15) Based On BLASTAnalysis Gen Bank Acession No. Species NP_014818.1 Saccharomycescerevisiae XP_001643411.1 Vanderwaltozyma polyspora DSM 70294XP_448977.1 Candida glabrata XP_455985.1 Kluyveromyces lactisNP_986937.1 Ashbya gossypii ATCC 10895 XP_001385654.2 Pichia stipitisCBS 6054 XP_001487052.1 Pichia guilliermondii ATCC 6260 EDK36331.2Pichia guilliermondii ATCC 6260 XP_001525914.1 Lodderomyces elongisporusNRRL YB-4239 XP_461358.1 Debaryomyces hansenii CBS767 XP_713184.1Candida albicans SC5314 XP_001645053.1 Vanderwaltozyma polyspora DSM70294 XP_505624.1 Yarrowia lipolytica XP_001805526.1 Phaeosphaerianodorum SN15 XP_001598340.1 Sclerotinia sclerotiorum 1980 XP_001907785.1Podospora anserina XP_001931658.1 Pyrenophora tritici-repentis Pt-1C-BFPXP_001560657.1 Botryotinia fuckeliana B05.10 XP_963006.1 Neurosporacrassa OR74A XP_364011.2 Magnaporthe grisea 70-15 XP_001209647.1Aspergillus terreus NIH2624 XP_001822945.1 Aspergillus oryzae RIB40XP_001257694.1 Neosartorya fischeri NRRL 181 XP_747591.2 Aspergillusfumigatus Af293 XP_001270060.1 Aspergillus clavatus NRRL 1 NP_596779.1Schizosaccharomyces pombe XP_001396584.1 Aspergillus nigerXP_001229385.1 Chaetomium globosum CBS 148.51 XP_001248887.1Coccidioides immitis RS XP_664134.1 Aspergillus nidulans FGSC A4XP_566668.1 Cryptococcus neoformans var. neoformans JEC21 XP_001839338.1Coprinopsis cinerea okayama 7#130 XP_757554.1 Ustilago maydis 521The yeast and fungal protein sequences of Table 24 were aligned usingDNASTAR. Multiple sequence alignments and percent identity calculationswere performed using the Clustal W method of alignment (supra).

More specifically, default parameters for multiple protein alignmentusing the Clustal W method of alignment correspond to: GAP PENALTY=10,GAP LENGTH PENALTY=0.2, Delay Divergent Seqs(%)=30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUBwith the ‘slow-accurate’ option. The resulting alignment was analyzed todetermine the presence or absence of the non-plant motifs for Ale1homologs, as identified in U.S. Pat. Appl. Pub. No. 2008-0145867-A1.Specifically, these include: M-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG (SEQ IDNO:102), RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:103),EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:21) and SAxWHGxxPGYxx-[T/F]-F (SEQ IDNO:104), wherein X encodes any amino acid residue. The H is residue inSEQ ID NO:104 has been reported to be a likely active site residuewithin the protein.

Only one motif, i.e., EX₁₁WNX₂-[T/V]-X₂W (SEQ ID NO:21), was completelyconserved in all 33 of the organisms aligned. The remainingM-[V/I]-[L/I]-xxK-[L/V/I]-xxxxxxDG (SEQ ID NO:102),RxKYYxxWxxx-[E/D]-[A/G]xxxxGxG-[F/Y]-xG (SEQ ID NO:103) andSAxWHGxxPGYxx-[T/F]-F (SEQ ID NO:104) motifs were only partiallyconserved. Thus, these motifs were appropriately truncated to fit with 0mismatch (i.e., SAxWHG [SEQ ID NO:20]), 1 mismatch (i.e., RxKYYxxW [SEQID NO:19]), or 2 mismatches (i.e., M(V/I)(L/I)xxK(LVI) [SEQ ID NO:18])for the purposes of the present methodologies.

1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase [“LPAAT”] Family Motifs:Analysis of the protein alignment comprising ScLPAAT (SEQ ID NO:18),MaLPAAT1 (SEQ ID NO:15) and YILPAAT1 (SEQ ID NO:17) revealed that the1-acyl-sn-glycerol-3-phosphate acyltransferase family motif EGTR (SEQ IDNO:20) was present in each of the LPAAT orthologs. On this basis,MaLPAAT1 was identified as a likely LPAAT, that was clearlydistinguishable from the Morteriella alpina LPAAT-like proteinsdisclosed in Intl. App. Pub. No. WO 2004/087902 (i.e., SEQ ID NOs:93 and95).

It is noteworthy that the EGTR (SEQ ID NO:20) motif, while lacking inthe LPCAT sequences in Intl. App. Pub. No. WO 2004/087902, is present inCeLPCAT (SEQ ID NO:2). It appears that other residues distinguish LPAATand LPCAT sequences in LPAAT-like proteins. One such residue could bethe extension of the EGTR (SEQ ID NO:20) motif. Specifically, whereasthe EGTR motif in ScLPAAT (SEQ ID NO:18), MaLPAAT1 (SEQ ID NO:15) andYILPAAT1 (SEQ ID NO:17) is immediately followed by a serine residue, theEGTR motif in CeLPCAT is immediately followed by an asparagine residue.In contrast, the two LPCATs in Intl. App. Pub. No. WO 2004/087902 have avaline substituted for the arginine residue in the EGTR motif and themotif is immediately followed by a valine residue.

Construction of pY201, Comprising a Codon-Optimized Saccharomycescerevisiae Ale1 Gene

The Saccharomyces cerevisiae ORF designated as “ScAle1” (SEQ ID NO:14)was optimized for expression in Yarrowia lipolytica, by DNA 2.0 (MenloPark, Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1cloning sites were introduced within the synthetic gene (i.e., ScAle1S;SEQ ID NO:22). None of the modifications in the ScAle1S gene changed theamino acid sequence of the encoded protein (i.e., the protein sequenceencoded by the codon-optimized gene [i.e., SEQ ID NO:23] is identical tothat of the wildtype protein sequence [i.e., SEQ ID NO:15]). ScAle1S wascloned into pJ201 (DNA 2.0) to result in pJ201:ScAle1S.

A 1863 by Pci1/Not1 fragment comprising ScAle1S was excised frompJ201:ScAle1S and used to create pY201 (SEQ ID NO:139; Table 25; FIG.10A). In addition to comprising a chimeric YAT1::ScAle1S::Lip1 gene,pY201 also contains a Y. lipolytica URA3 selection marker flanked byLoxP sites for subsequent removal, if needed, by Crerecombinase-mediated recombination. Both the YAT1::ScAle1S::Lip1chimeric gene and the URA3 gene were flanked by fragments havinghomology to 5′ and 3′ regions of the Y. lipolytica Pox3 gene tofacilitate integration by double homologous recombination, althoughintegration into Y. lipolytica is known to usually occur withouthomologous recombination. Thus, construct pY201 thereby contained thefollowing components:

TABLE 25 Description of Plasmid pY201 (SEQ ID NO: 139) RE Sites AndNucleotides Within SEQ ID NO: 139 Description Of Fragment And ChimericGene Components BsiW1/Sbf1 LoxP::Ura3::LoxP, comprising: (1-1706 bp)LoxP sequence (SEQ ID NO: 140) Yarrowia lipolytica Ura3 gene (GenBankAccession No. AJ306421); LoxP sequence (SEQ ID NO: 140) Sbf1/Sph1 3′portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3 (1706-3043 bp)(GenBank Accession No. YALI0D24750g) (i.e., bp 2215-3038 in pY201)Sph1/Asc1 ColE1 plasmid origin of replication; (3043-5743 bp)Ampicillin-resistance gene (Amp^(R)) for selection in E. coli (i.e., bp3598-4758 [complementary] in pY201); E. coli f1 origin of replicationAscI/BsiWI 5′ portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3(5743-6513 bp) (GenBank Accession No. YALI0D24750g) (i.e., bp 5743-6512in pY201) BsiWI/BsiWI YAT1::ScAle1S::Lip1, comprising: (6514-1 bp) YAT1:Yarrowia lipolytica YAT1 promoter (U.S. Pat. Appl. Pub. [a Not1 site,located No. 2006/0094102-A1) (i.e., bp 6514-7291 in pY201) betweenScAle1S ScAle1S: codon-optimized Ale1 (SEQ ID NO: 22) derived from andLip1 is present Saccharomyces cerevisiae YOR175C (i.e., bp 7292-9151 inat bp pY201; labeled as “Sc LPCATs ORF” in Figure); 9154 bp] Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) (i.e., bp 9160-9481 pY201; labeled as “Lip1-3′” in Figure)Construction of pY168, Comprising a Yarrowia lipolytica Ale1 Gene

The Yarrowia lipolytica ORF designated as “YIAle1” (GenBank AccessionNo. XP 505624; SEQ ID NO:16) was amplified by PCR from Yarrowialipolytica ATCC #20362 cDNA library using PCR primers 798 and 799 (SEQID NOs:141 and 142, respectively). Additionally, the YAT promoter wasamplified by PCR primers 800 and 801 (SEQ ID NOs:143 and 144,respectively) from pY201 (SEQ ID NO:139). Since the primer pairs weredesigned to create two PCR products having some overlap with oneanother, a YAT1::YIAle1 fusion fragment was then amplified byoverlapping PCR using primers 798 and 801 (SEQ ID NOs:141 and 144,respectively) and the two PCR fragments as template. The PCR was carriedout in a RoboCycler Gradient 40 PCR machine (Stratagene) using themanufacturer's recommendations and Pfu Ultra™ High-Fidelity DNAPolymerase (Stratagene, Cat. No. 600380). Amplification was carried outas follows: initial denaturation at 95° C. for 4 min, followed by 30cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 1min, and elongation at 72° C. for 1 min. A final elongation cycle at 72°C. for 10 min was carried out, followed by reaction termination at 4° C.

The PCR product comprising the YAT1::YI Ale1 fusion fragment was gelpurified and digested with ClaI/NotI. This Cla1-Not1 fragment wasligated into pY201 that had been similarly digested (thereby removingthe YAT1::ScAle1S fragment) to create pY168 (SEQ ID NO:145), comprisinga chimeric YAT1::YIAle1::Lip1 gene. The DNA sequence of the YarrowiaAle10RF was confirmed by DNA sequencing. The components present in pY168(FIG. 10B; SEQ ID NO:145) are identical to those present in pY201, withthe exception of the YAT1::YIAle1::Lip1 gene in pY168, instead of theYAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A). Note that YIAle1 islabeled as “YI LPCAT” in FIG. 10B.

Construction of pY208, Comprising a Mortierella alpina LPAAT1 Gene

The Mortierella alpina ORF designated as “MaLPAAT1” (SEQ ID NO:28) wasoptimized for expression in Yarrowia lipolytica, by DNA 2.0 (Menlo Park,Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1 cloningsites were introduced within the synthetic gene (i.e., MaLPAAT1S; SEQ IDNO:35). None of the modifications in the MaLPAAT1S gene changed theamino acid sequence of the encoded protein (i.e., the protein sequenceencoded by the codon-optimized gene [i.e., SEQ ID NO:36] is identical tothat of the wildtype protein sequence [i.e., SEQ ID NO:29]). MaLPAAT1 Swas cloned into pJ201 (DNA 2.0) to result in pJ201:MaLPAAT1S.

A 945 by Pci1/Not1 fragment comprising MaLPAAT1S was excised frompJ201:MaLPAAT1S and used to create pY208 (SEQ ID NO:146), in a 3-wayligation with two fragments of pY201 (SEQ ID NO:139). Specifically, theMaLPAAT1 fragment was ligated with a 3530 by Sph-NotI pY201 fragment anda 4248 by NcoI-SphI pY201 fragment to result in pY208. The componentspresent in pY208 (FIG. 11A; SEQ ID NO:146) are identical to thosepresent in pY201, with the exception of the YAT1::MaLPAAT1S::Lip1 genein pY208, instead of the YAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A).

Construction of pY207, Comprising a Yarrowia lipolytica LPAAT1 Gene

A putative LPAAT1 from Yarrowia lipolytica (designated herein as“YILPAAT1”; SEQ ID NOs:30 and 31) was described in U.S. Pat. No.7,189,559 and GenBank Accession No. XP_(—)504127. The protein isannotated as “similar to uniprot|P33333 Saccharomyces cerevisiae YDL052cSLC1 fatty acyltransferase”.

The YILPAAT10RF (SEQ ID NO:30) was amplified by PCR using a Yarrowialipolytica ATCC #20362 cDNA library as a template and PCR primers 856and 857 (SEQ ID NOs:147 and 148, respectively). The PCR was conductedusing the same components and conditions as described above foramplification of the YAT1::YI Ale1 fusion fragment, prior to synthesisof pY168.

The PCR product comprising the YILPAAT10RF was digested with PciI andNotI and then utilized in a 3-way ligation with two fragments frompY168. Specifically, the YILPAAT1 fragment was ligated with a 3530 bySph-NotI pY168 fragment and a 4248 by NcoI-SphI pY168 fragment, toproduce pY207, comprising a chimeric YAT1::YILPAAT1::Lip1 gene. The Y.lipolytica LPAAT10RF was confirmed by DNA sequencing. The componentspresent in pY207 (FIG. 11B; SEQ ID NO:149) are identical to thosepresent in pY201, with the exception of the chimericYAT1::YILPAAT1::Lip1 gene in pY207, instead of the YAT1::ScAle1S::Lip1gene in pY201 (FIG. 10A). Note that YILPAAT1 is labeled as “YI LPAT1ORF” in FIG. 11B.

Construction of pY175, Comprising a Caenorhabditis elegans LPCAT Gene

The Caenorhabditis elegans ORF designated as “CeLPCAT” (SEQ ID NO:24)was optimized for expression in Yarrowia lipolytica, by GenScriptCorporation (Piscataway, N.J.). In addition to codon optimization, 5′Nco1 and 3′ Not1 cloning sites were introduced within the synthetic gene(i.e., CeLPCATS; SEQ ID NO:26). None of the modifications in theCeLPCATS gene changed the amino acid sequence of the encoded protein(i.e., the protein sequence encoded by the codon-optimized gene [i.e.,SEQ ID NO:27] is identical to that of the wildtype protein sequence[i.e., SEQ ID NO:25]).

A Nco1-Not1 fragment comprising CeLPCATS was used to create pY175 (SEQID NO:150), in a 3-way ligation with two fragments from pY168 (SEQ IDNO:145). Specifically, the Nco1-Not1 fragment comprising CeLPCATS wasligated with a 3530 by Sph-NotI pY168 fragment and a 4248 by NcoI-SphIpY168 fragment to result in pY175. The components present in pY175 (FIG.12A; SEQ ID NO:150) are identical to those present in pY201, with theexception of the YAT1::CeLPCATS::Lip1 gene in pY175, instead of theYAT1::ScAle1S::Lip1 gene in pY201 (FIG. 10A). Note that CeLPCATS islabeled as “Ce.LPCATsyn” in FIG. 12A.

Example 9 Functional Characterization of Different LPLATs inEPA-Producing

Yarrowia lipolytica Strain Y8406

Yarrowia lipolytica strain Y8406U, producing EPA, was used tofunctionally characterize the effects of overexpression of theSaccharomyces cerevisiae Ale1, Yarrowia lipolytica Ale1, Mortierellaalpina LPAAT1, Yarrowia lipolytica LPAAT1 and Caenorhabditis elegansLPCAT, following their stable integration into the Yarrowia hostchromosome. This was in spite of the host containing its native LPLATs,i.e., Ale1 and LPAAT1.

Transformation and Growth

To disrupt the Ura3 gene, construct pZKUM (FIG. 5A; SEQ ID NO:130;described in Table 15 of U.S. Pat. Appl. Pub. No. 2009-0093543-A1) wasused to integrate a Ura3 mutant gene into the Ura3 gene of strain Y8406in a manner similar to that described for pZKUM transformation of strainY8006 (Example 3). Several transformants were grown and identified topossess a Ura− phenotype.

GC analyses showed that there were 26.1% EPA of FAMEs inpZKUM-transformant strains #4 and #5. These two strains were designatedas strains Y8406U1 and Y8406U2, respectively (collectively, Y8406U).

Yarrowia lipolytica strain Y8406U was then individually transformed withlinear SphI-AscI fragments of the integrating vectors described inExample 8, wherein each LPLAT was under the control of the Yarrowia YAT1promoter. Specifically, vectors pY201 (YAT1::ScAle1S::Lip1), pY168(YAT1::YIAle1::Lip1), pY208 (YAT1::MaLPAAT1S::Lip1), pY207(YAT1::YILPAAT1::Lip1) and pY175 (YAT1::CeLPCATS::Lip1) were transformedaccording to the General Methods.

Each transformation mix was plated on MM agar plates. Several resultantURA+ transformants were picked and inoculated into 3 mL FM medium(Biomyx Cat. No. CM-6681, Biomyx Technology, San Diego, Calif.)containing per L: 6.7 g Difco Yeast Nitrogen Base without amino acids, 5g Yeast Extract, 6g KH₂PO₄, 2g K₂HPO₄, 1.5 g MgSO₄.7H₂O, 1.5 mgthiamine.HCl, and 20g glucose. After 2 days growth on a shaker at 200rpm and 30° C., the cultures were harvested by centrifugation andresuspended in 3 mL HGM medium (Cat. No. 2G2080, Teknova Inc.,Hollister, Calif.) containing 0.63% monopotassium phosphate, 2.7%dipotassium phosphate, 8.0% glucose, adjusted to pH 7.5. After 5 daysgrowth on a shaker at 200 rpm and at 30° C., 1 mL aliquots of thecultures were harvested by centrifugation and analyzed by GC.Specifically, the cultured cells were collected by centrifugation for 1min at 13,000 rpm, total lipids were extracted, and fatty acid methylesters [“FAMEs”] were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC (General Methods).

Based on the fatty acid composition of the 3 mL cultures, selectedtransformants were further characterized. Specifically, clones #5 and#11 of strain Y8406U transformed with expression vector pY201(comprising ScAle1S) were selected and designated as “Y8406U::ScAle1S-5”and “Y8406U::ScAle1S-11”, respectively; clone #16 of strain Y8406Utransformed with expression vector pY168 (comprising YIAle1) wasselected and designated as “Y8406U::YIAle1”; clone #8 of strain Y8406Utransformed with expression vector pY208 (comprising MaLPAAT1 S) wasselected and designated as “Y8406U::MaLPAAT1S”; clone #21 of strainY8406U transformed with expression vector pY207 (comprising YILPAAT1)was selected and designated as “Y8406U::YILPAAT1”; and clone #23 ofstrain Y8406U transformed with expression vector pY175 (comprisingCeLPCATS) was selected and designated as “Y8406U::CeLPCATS”.Additionally, strain Y8406 (a Ura+ strain that was parent to strainY8406U (Ura−)) was used as a control.

Each selected transformant and the control was streaked onto MM agarplates. Then, one loop of freshly streaked cells was inoculated into 3mL FM medium and grown overnight at 250 rpm and 30° C. The OD_(600nm)was measured and an aliquot of the cells were added to a finalOD_(600nm) of 0.3 in 25 mL FM medium in a 125 mL flask. After 2 days ina shaker incubator at 250 rpm and at 30° C., 6 mL of the culture washarvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for GC analysis (supra) and 10 mL dried for dry cellweight [“DCW”] determination.

For DCW determination, 10 mL culture was harvested by centrifugation for5 min at 4000 rpm in a Beckman GH-3.8 rotor in a Beckman GS-6Rcentrifuge. The pellet was resuspended in 25 mL of water andre-harvested as above. The washed pellet was re-suspended in 20 mL ofwater and transferred to a pre-weighed aluminum pan. The cell suspensionwas dried overnight in a vacuum oven at 80° C. The weight of the cellswas determined.

Lipid Content, Fatty Acid Composition and Conversion Efficiencies

A total of four separate experiments were conducted under identicalconditions. Experiment 1 compared control strain Y8406 versus strainY8406U::ScAle1S-5. Experiment 2 compared control strain Y8406 versusstrain Y8406U::YIAle1. Experiment 3 compared control strain Y8406 versusstrain Y8406U::YIAle1, strain Y8406U::ScAle1S-11, and strainY8406U::MaLPAAT1S. Experiment 4 compared control strain Y8406 versusstrain Y8406U::MaLPAAT1S, strain Y8406U::YILPAAT1 and strainY8406U::CeLPCATS.

In each experiment, the lipid content, fatty acid composition and EPA asa percent of the DCW are quantified for 1, 2 or 3 replicate cultures[“Replicates”] of the control Y8406 strain and the transformant Y8406Ustrain(s). Additionally, data for each Y8406U transformant is presentedas a % of the Y8406 control. Table 26 below summarizes the total lipidcontent of cells [“TFAs % DCW”], the concentration of each fatty acid asa weight percent of TFAs [“% TFAs”] and the EPA content as a percent ofthe dry cell weight [“EPA % DCW”]. More specifically, fatty acids areidentified as 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0 (stearicacid), 18:1 (oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA, ETrA, ETA andEPA.

Table 27 summarizes the conversion efficiency of each desaturase and theΔ9 elongase functioning in the PUFA biosynthetic pathway and which arerequired for EPA production. Specifically, the Δ12 desaturase conversionefficiency [“Δ12 CE”], Δ8 desaturase conversion efficiency [“Δ8 CE”], Δ5desaturase conversion efficiency [“Δ5 CE”], Δ17 desaturase conversionefficiency [“Δ17 CE”] and Δ9 elongation conversion efficiency [“Δ9e CE”]are provided for each control Y8406 strain and the transformant Y8406Ustrain(s); data for each Y8406U transformant is presented as a % of theY8406 control. Conversion efficiency was calculated according to theformula: product(s)/(product(s)+substrate)*100, where product includesboth product and product derivatives.

TABLE 26 Lipid Content And Composition In LPCAT Transformant Strains OfYarrowia lipolytica Y8406 TFA EPA % % TFAs % Expt. Strain Replicates DCW16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA ERA ETA EPA DCW 1 Y8406 AVG.317.6 3.8 0.7 3.3 6.4 22.6 2.5 2.8 2.2 0.5 1.9 2.0 48.9 8.6 Y8406U::AVG.3 18.3 4.2 0.7 3.5 5.7 15.1 0.6 3.3 3.7 0.8 1.8 2.3 56.9 10.4ScAle1S-5 % Ctrl 104 111 100 106 89 67 24 118 168 160 95 115 116 121 2Y8406 AVG.3 23.2 3.5 0.6 3.3 6.4 22.3 2.7 2.6 2.1 0.5 1.6 2.0 49.9 11.6Y8406U:: AVG.3 22.3 3.8 0.7 2.9 3.9 12.7 0.4 3.0 3.8 0.8 1.6 2.4 60.913.6 YIAle1 % Ctrl 96 109 117 88 61 57 15 115 181 160 100 120 122 117 3Y8406 1 26.1 2.7 0.7 2.8 6.5 20.5 2.5 3.2 2.3 0.7 0.8 0.0 50.8 13.3Y8406U:: AVG.2 23.3 3.3 0.7 2.4 3.6 12.1 0.5 3.2 3.5 0.9 0.0 2.3 62.214.5 YIAle1 % Ctrl 89 122 100 86 55 59 20 100 152 129 0 na 122 109Y8406U:: AVG.2 28.0 3.0 0.7 3.0 5.5 13.1 0.6 3.5 3.8 0.9 0.0 2.4 58.516.4 ScAle1S-11 % Ctrl 107 111 100 107 85 64 24 109 165 129 0 na 115 123Y8406U:: AVG.2 23.7 4.4 0.8 4.2 6.6 11.2 0.7 2.7 3.7 0.9 0.0 2.5 57.013.5 MaLPAAT1S % Ctrl 91 163 114 150 102 55 28 84 161 129 0 na 112 102 4Y8406 AVG.2 27.9 2.8 0.6 3.1 6.2 20.6 2.9 2.9 2.0 0.6 0.7 2.0 49.4 13.8Y8406U:: AVG.2 25.2 4.8 0.8 4.8 6.9 11.6 0.8 2.5 3.0 0.7 0.0 2.3 55.314.0 MaLPAAT1S % Ctrl 90 171 133 155 111 56 28 86 150 117 0 115 112 101Y8406U:: AVG.2 25.2 3.7 0.7 4.2 6.2 13.0 1.2 2.3 2.6 0.6 0.0 2.2 56.714.3 YILPAAT1 % Ctrl 90 132 117 135 100 63 41 79 130 100 0 110 115 104Y8406U:: AVG.2 24.7 3.8 0.6 4.6 7.1 13.9 1.6 2.3 2.6 0.6 0.4 2.2 53.613.2 CeLPCATS % Ctrl 89 136 100 148 115 67 55 79 130 100 57 110 109 96

TABLE 27 Desaturase And Elongase Conversion Efficiency In LPCATTransformant Strains Of Yarrowia lipolytica Y8406 Expt. StrainReplicates Δ12 CE Δ9e CE Δ8 CE Δ5 CE Δ17 CE 1 Y8406 AVG.3 93 70 92 92 90Y8406U::ScAle1S-5 AVG.3 94 81 93 91 89 % Ctrl 101 116 101 98 98 2 Y8406AVG.3 93 70 93 93 91 Y8406U::YIAle1 AVG.3 96 85 94 91 90 % Ctrl 103 121101 98 98 3 Y8406 1 93 72 93 96 89 Y8406U::YIAle1 AVG.2 96 85 96 92 89 %Ctrl 104 119 103 96 100 Y8406U::ScAle1S-11 AVG.2 94 83 95 91 88 % Ctrl101 117 102 95 99 Y8406U::MaLPAAT1S AVG.2 92 85 96 90 89 % Ctrl 100 119103 94 100 4 Y8406 AVG.2 93 71 94 93 91 Y8406U::MaLPAAT1S AVG.2 92 84 9691 90 % Ctrl 99 118 102 99 100 Y8406U::YILPAAT1 AVG.2 93 82 96 92 92 %Ctrl 100 115 103 100 101 Y8406U::CeLPCATS AVG.2 92 80 96 92 91 % Ctrl 99113 102 99 100

Based on the data concerning Experiments 1, 2 and 3 in Table 26 andTable 27, overexpression of LPLAT in EPA strains Y8406U::ScAle1S-5,Y8406U::ScAle1S-11, Y8406U::YIAle1 and Y8406U::MaLPAAT1S results insignificant reduction (to 67% or below of the control) of theconcentration of LA (18:2) as a weight % of TFAs [“LA % TFAs”], anincrease (to at least 12% of the control) in the concentration of EPA asa weight % of TFAs [“EPA % TFAs”], and an increase (to at least 16% ofthe control) in the conversion efficiency of the Δ9 elongase. Comparedto Y8406U::ScAle1S-5 and Y8406U::ScAle1S-11, Y8406U::YIAle1 has lower LA% TFAs, higher EPA % TFAs, better Δ9 elongation conversion efficiency,and slightly lower TFAs % DCW and EPA % DCW. Y8406U::YIAle1 andY8406U::MaLPAAT1S are similar except overexpression of MaLPAAT1 Sresulted in lower LA % TFAs, EPA % TFAs, and EPA % DCW.

Experiment 4 shows that overexpression of LPLAT in EPA strainsY8406U::YIAle1, Y8406U::MaLPAAT1S and Y8406U::CeLPCATS results insignificant reduction (to 67% or below of the control) of LA % TFAs, anincrease (to at least 9% of the control) in EPA % TFAs, and an increase(to at least 13% of the control) in the conversion efficiency of the Δ9elongase. Compared to Y8406U::CeLPCATS, Y8406U::YLPAAT1 andY8406U::MaLPAAT1S both have lower LA % TFAs, higher EPA % TFAs, higherEPA % DCW, and slightly better TFAs % DCW. Y8406U::YILPAAT1 andY8406U::MaLPAAT1S are similar except overexpression of MaLPAAT1S resultsin lower LA % TFAs, slightly lower EPA % TFAs and EPA % DCW, andslightly better Δ9 elongase conversion efficiency.

It is well known in the art that most desaturations occur at the sn-2position of phospholipids, while fatty acid elongations occur onacyl-CoAs. Furthermore, ScAle1S, YIAle1, MaLPAAT1S and YILPAAT1 wereexpected to only incorporate acyl groups from the acyl-CoA pool into thesn-2 position of lysophospholipids, such as lysophosphatidic acid[“LPA”] and lysophosphatidylcholine [“LPC”]. Thus, it was expected thatexpression of ScAle1S, YIAle1, MaLPAAT1S, and YILPAAT1 would result inimproved desaturations due to improved substrate availability inphospholipids, and not result in improved elongations that requireimproved substrate availability in the CoA pool. Our data (supra) showsthat unexpectedly, expression of ScAle1S, YIAle1, MaLPAAT1S and YILPAAT1significantly improved the Δ9 elongase conversion efficiency in strainsof Yarrowia producing EPA but did not improve the desaturations(measured as Δ12 desaturase conversion efficiency, Δ8 desaturaseconversion efficiency, Δ5 desaturase conversion efficiency or Δ17desaturase conversion efficiency).

CeLPCAT was previously shown to improve Δ6 elongation conversionefficiency in Saccharomyces cerevisiae fed LA or GLA (Intl App. Pub. No.WO 2004/076617). This was attributed to its reversible LPCAT activitythat released fatty acids from phospholipids into the CoA pool. Animprovement in Δ9 elongation conversion efficiency in an oleaginousmicrobe, such as Yarrowia lipolytica, engineered for high level LC-PUFAproduction in the absence of feeding fatty acids was not contemplated inIntl. App. Pub. No. WO 2004/076617.

Furthermore, expression of ScAle1S, YIAle1, MaLPAAT1S, YILPAAT1 andCeLPCATS did not significantly alter either the level of PUFAsaccumulated or the total lipid content in strains of Yarrowia producingEPA. Previous studies have shown that both Δ6 elongation and Δ9elongation are bottlenecks in long chain PUFA biosynthesis due to poortransfer of acyl groups between phospholipid and acyl-CoA pools. Basedon the improved Δ9 elongase conversion efficiency resulting fromover-expression of LPLATs, demonstrated above, it is anticipated thatthe LPLATs described herein and their orthologs, such as ScLPAAT, willalso improve Δ6 elongation conversion efficiency.

Example 10 Construction of Expression Vectors Comprising LPAAT ORFs andan Autonomously Replicating Sequence

The present example describes the construction of vectors comprisingautonomously replicating sequences [“ARS”] and LPAAT ORFs suitable forLPAAT gene expression without integration in Yarrowia lipolytica. ORFsincluded the Saccharomyces cerevisiae LPAAT encoding SEQ ID NO:32 andthe Yarrowia lipolytica LPAAT1 encoding SEQ ID NO:31. Example 11describes the results obtained following transformation of these vectorsinto Y. lipolytica.

Construction of pY222, Comprising a Codon-Optimized Saccharomycescerevisiae LPAAT Gene

The Saccharomyces cerevisiae ORF designated as “ScLPAAT” (SEQ ID NO:32)was optimized for expression in Yarrowia lipolytica, by DNA 2.0 (MenloPark, Calif.). In addition to codon optimization, 5′ Pci1 and 3′ Not1cloning sites were introduced within the synthetic gene (i.e., ScLPAATS;SEQ ID NO:151). None of the modifications in the ScLPAATS gene changedthe amino acid sequence of the encoded protein (i.e., the proteinsequence encoded by the codon-optimized gene [i.e., SEQ ID NO:152] isidentical to that of the wildtype protein sequence [i.e., SEQ IDNO:32]). ScLPAATS was cloned into pJ201 (DNA 2.0) to result inpJ201:ScLPAATS.

A 926 by Pci11Not1 fragment comprising ScLPAATS was excised frompJ201:ScLPAATS and cloned into NcoI-Not1 cut pYAT-DG2-1 to create pY222(SEQ ID NO:153; Table 28; FIG. 14A). Thus, pY222 contained the followingcomponents:

TABLE 28 Description of Plasmid pY222 (SEQ ID NO: 153) RE Sites AndNucleotides Within SEQ ID NO: 153 Description Of Fragment And ChimericGene Components Sa/1/SwaI YAT1::ScLPAATS::Lip1, comprising: (1-2032)YAT1: Yarrowia lipolytica YAT1 promoter (U.S. Pat. Appl. Pub. No.2006/0094102-A1); ScLPAATS: codon-optimized ScLPAATS (SEQ ID NO: 151)(labeled as “Sc LPAATs ORF” in Figure); Lip1: Lip1 terminator sequencefrom Yarrowia Lip1 gene (GenBank Accession No. Z50020) (labeled as“Lip1-3′” in Figure) SwaI/AvaI CoIE1 plasmid origin of replication;(2032-4946) Ampicillin-resistance gene (Amp^(R)) for selection in E.coli; E. coli f1 origin of replication AvaI-SphI Yarrowia lipolyticacentromere and autonomously replicating (4946-6330) sequence [“ARS”] 18locus SphI-Sa/I Yarrowia lipolytica URA3 gene (GenBank Accession No.(6330-1) AJ306421)Construction of pY177, Comprising a Yarrowia lipolytica LPAAT1 Gene

The Yarrowia lipolytica centromere and autonomously replicating sequence[“ARS”] was amplified by standard PCR using primer 869 (SEQ ID NO:154)and primer 870 (SEQ ID NO:155), with plasmid pYAT-DG2-1 as template. ThePCR product was digested with AscI/AvrII and cloned into AscI-AvrIIdigested pY207 (SEQ ID NO:149; Example 8) to create pY177 (SEQ IDNO:156; Table 29; FIG. 14B). Thus, the components present in pY177 areidentical to those in pY207 (FIG. 12A), except for the replacement ofthe 373 by pY207 sequence between AscI and AvrII with the 1341 bysequence containing ARS. More specifically, pY177 contained thefollowing components:

TABLE 29 Description of Plasmid pY177 (SEQ ID NO: 156) RE Sites AndNucleotides Within SEQ ID NO: 156 Description Of Fragment And ChimericGene Components BsiW1/Sbf1 LoxP::Ura3::LoxP, comprising: (1-1706 bp)LoxP sequence (SEQ ID NO: 140) Yarrowia lipolytica Ura3 gene (GenBankAccession No. AJ306421); LoxP sequence (SEQ ID NO: 140) Sbf1/Sph1 3′portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3 (1706-3043 bp)(GenBank Accession No. YALI0D24750g) SphI/AscI ColE1 plasmid origin ofreplication; (3043-5743 bp) Ampicillin-resistance gene (Amp^(R)) forselection in E. coli; E. coli f1 origin of replication AscI/BsiWI 5′portion of Yarrowia lipolytica POX3 Acyl-CoA oxidase 3 (5743-6513 bp)(GenBank Accession No. YALI0D24750g) AscI/AvrII Yarrowia lipolyticacentromere and autonomously replicating (5743-7084 bp) sequence [“ARS”]18 locus AvrII/BsiWI 5′ portion of Yarrowia lipolytica POX3 Acyl-CoAoxidase 3 (7084-7481 bp) (GenBank Accession No. YALI0D24750g)BsiWI/BsiWI YAT1::YILPAAT1::Lip1, comprising: (7481-1 bp) YAT1: Yarrowialipolytica YAT1 promoter (U.S. Pat. Appl. Pub. No. 2006/0094102-A1);YILPAAT1: Yarrowia lipolytica LPAAT1 (“YALI0E18964g”; GenBank AccessionNo. XP_504127) (SEQ ID NO: 30) (labeled as “YI LPAT1 ORF” in Figure);Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBankAccession No. Z50020) (labeled as “Lip1-3′” in Figure)

Example 11 Functional Characterization of Different LPAATs inEPA-Producing Yarrowia lipolytica Strain Y8406

Yarrowia lipolytica strain Y8406U, producing EPA, was used tofunctionally characterize the effects of expression of the Saccharomycescerevisiae LPAATS (SEQ ID NO:151) and Yarrowia lipolytica LPAAT1 (SEQ IDNO:30) without integration on self-replicating plasmids. This was inspite of the host containing its native LPAATs.

Transformation and Growth

Yarrowia lipolytica strain Y8406U (Example 9) was individuallytransformed with uncut plasmids from Example 10. Specifically, vectorspY177 (YAT1::YILPAAT1::Lip1) [SEQ ID NO:156] and pY222(YAT1::ScLPAATS::Lip1) [SEQ ID NO:153] were transformed according to theGeneral Methods.

Each transformation mix was plated on MM agar plates. Several resultantURA+ transformants were picked and inoculated into 3 mL CSM-U medium(Teknova Cat. No. C8140, Teknova Inc., Hollister, Calif.), wherein CSM-Umedium refers to CM Broth with glucose minus uracil containing 0.13%amino acid dropout powder minus uracil, 0.17% yeast nitrogen base, 0.5%(NH₄)₂SO₄, and 2.0% glucose. After 2 days growth on a shaker at 200 rpmand 30° C., the cultures were harvested by centrifugation andresuspended in 3 mL HGM medium (Cat. No. 2G2080, Teknova Inc.). After 5days growth on a shaker, 1 mL aliquots of the cultures were harvestedand analyzed by GC, as described in Example 9.

Based on the fatty acid composition of the 3 mL cultures, selectedtransformants were further characterized by flask assay. Specifically,clones #5 and #6 of strain Y8406U transformed with expression vectorpY222 (comprising ScLPAATS) were selected and designated as“Y8406U::ScLPAATS-5” and “Y8406U::ScLPAATS-6”, respectively; clone #1 ofstrain Y8406U transformed with expression vector pY177 (comprisingYILPAAT1) was selected and designated as “Y8406U::YILPAAT1”.Additionally, strain Y8406 (a Ura+strain that was parent to strainY8406U (Ura−)) was used as a control.

Each selected transformant and the control was streaked onto MM agarplates. Then, one loop of freshly streaked cells was inoculated into 3mL CSM-U medium and grown overnight at 250 rpm and 30° C. The OD_(600nm)was measured and an aliquot of the cells were added to a finalOD_(600nm) of 0.3 in 25 mL CSM-U medium in a 125 mL flask. After 2 daysin a shaker incubator at 250 rpm and at 30° C., 6 mL of the culture washarvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for GC analysis and 10 mL dried for dry cell weight[“DCW”] determination, as described in Example 9.

Lipid Content, Fatty Acid Composition and Conversion Efficiencies

The lipid content, fatty acid composition and EPA as a percent of theDCW are quantified for 2 replicate cultures [“Replicates”] of thecontrol Y8406 strain and the transformant Y8406U strain(s).Additionally, data for each Y8406U transformant is presented as a % ofthe Y8406 control. Table 30 below summarizes the total lipid content ofcells [“TFAs % DCW”], the concentration of each fatty acid as a weightpercent of TFAs [“% TFAs”] and the EPA content as a percent of the drycell weight [“EPA % DCW”]. More specifically, fatty acids are identifiedas 16:0 (palmitate), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1(oleic acid), 18:2 (LA), ALA, EDA, DGLA, ARA, ETrA, ETA and EPA.

Table 31 summarizes the conversion efficiency of each desaturase and theΔ9 elongase functioning in the PUFA biosynthetic pathway and which arerequired for EPA production, in a manner identical to that described inExample 9.

TABLE 30 Lipid Content And Composition In ScLPAATS and YILPAAT1Transformant Strains Of Yarrowia lipolytica Y8406 TFA % % TFAs EPA %Strain Replicates DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA ERA ETAEPA DCW Y8406 AVG.2 22.0 2 0 2 4 19 2 3 4 1 2 3 55 12 Y8406U:: AVG.224.6 2 1 2 6 14 1 3 5 1 2 3 55 14 YILPAAT1 % Ctrl 112 98 153 102 148 7650 120 144 101 109 123 101 113 Y8406U:: AVG.2 21.6 3 1 3 6 14 1 3 4 1 23 57 12 ScLPAATS-5 % Ctrl 98 131 137 125 131 74 56 100 117 86 101 108104 102 Y8406U:: AVG.2 21.4 3 1 3 5 14 1 3 4 1 2 3 58 12 ScLPAATS-6 %Ctrl 97 125 133 121 124 72 52 97 119 88 102 111 106 103

TABLE 31 Desaturase And Elongase Conversion Efficiency In ScLPAATS andYILPAAT1 Transformant Strains Of Yarrowia lipolytica Y8406 Δ9e Δ8 Δ5 Δ17Strain Replicates Δ12 CE CE CE CE CE Y8406 AVG.2 95 77 92 90 92Y8406U::YILPAAT1 AVG.2 93 82 92 87 90 % Ctrl 98 107 99 97 98Y8406U::ScLPAATS-5 AVG.2 94 83 93 89 92 % Ctrl 98 108 100 99 100Y8406U::ScLPAATS-6 AVG.2 94 83 93 89 92 % Ctrl 99 109 101 99 100

Based on the data in Table 30 and Table 31 above, overexpression of bothScLPAATS and YILPAAT1 in EPA strains Y8406U::YILPAAT1,Y8406U::ScLPAATS-5 and Y8406U::ScLPAATS-6 resulted in reduction (to 76%or below of the control) of the concentration of LA (18:2) as a weight %of TFAs [“LA % TFAs”], and an increase (to at least 7% of the control)in the conversion efficiency of the Δ9 elongase. ScLPAATS and YILPAAT1have a similar effect on lipid profile.

The results obtained above were then compared to those obtained inExample 9, although different means were utilized to characterize theLPLATs. Specifically, in Example 9, linearized DNA carrying the LPLATswere transformed by chromosomal integration, since the vectors lackedARS sequences. This resulted in stable integrations and the strains weregrown in the relatively rich, non-selective FM growth medium during bothpreculture and 2 days growth prior to being transferred to HGM.

In Example 11, the functional characterization of YILPAAT1 and ScLPAATSwas done on a replicating plasmid. Thus, Yarrowia lipolytica strainY8406 was transformed with circular DNA carrying each LPAAT and ARSsequence. To maintain these plasmids and assay gene expression withoutintegration, it was necessary to grow the transformants on selectivemedium (i.e., CSM-U medium) during both preculture and 2 days growthprior to being transferred to HGM.

These differences described above can contribute to differences in lipidprofile and content, as illustrated by the expression of YILPAAT1 inExamples 9 and 11. The change over control in LA % TFAs, EPA % TFAs, andΔ9 elongase conversion efficiency were 63%, 115%, and 115%,respectively, upon expression of YILPAAT in Example 9, whereas thechange over control in LA % TFAs, EPA % TFAs, and Δ9 elongase conversionefficiency were 76%, 101%, and 107%, respectively, upon expression ofYILPAAT in Example 11. Thus, the improvements in Δ9 elongation andLC-PUFA biosynthesis in Example 11 are minimized when compared to thoseobserved in Example 9. These differences can be attributed to the“position effects” of chromosomal integration and/or different growthconditions.

Since the improvements in LC-PUFA biosynthesis (measured as reduction inLA % TFAs, increase in EPA % TFAs and increase in Δ9 elongase conversionefficiency) are similar for both ScLPAATS and YILPAAT when transformedin Yarrowia lipolytica strain Y8406 on a replicating plasmid, it isanticipated that both LPLAATs will also function similarly when stablyintegrated into the host chromosome. Thus, ScLPAATS will likely improvethe lipid profile in a manner similar to that observed in Example 9,when YILPAAT1 was stably integrated into the host chromosome.

1. An extracted oil comprising: (a) at least 50 weight percent ofeicosapentaenoic acid measured as a weight percent of total fatty acids;and, (b) having a ratio of at least 3.1 of eicosapentaenoic acid,measured as a weight percent of total fatty acids, to linoleic acid,measured as a weight percent of total fatty acids.
 2. The extracted oilof claim 1 wherein said oil is a microbial oil.
 3. The extracted oil ofclaim 2 wherein said oil is extracted from fermented recombinantYarrowia sp. cells, engineered for the production of eicosapentaenoicacid wherein said cells comprise: a) at least one multizyme whichcomprises a polypeptide having at least one Δ9 elongase linked to atleast one Δ8 desaturase; (b) at least one peroxisome biogenesis factorprotein whose expression has been down-regulated; and, (c) at least onerecombinant construct comprising a nucleotide sequence encoding anenzyme selected from the group consisting of malonyl CoA synthetase andacyl-CoA lysophospholipid acyltransferase.
 4. The extracted oil of claim3 wherein said oil is extracted from fermented recombinant Yarrowia sp.cells and wherein said cells comprise at least one recombinant constructcomprising a nucleotide sequence encoding malonyl CoA synthetase,wherein the malonyl CoA synthetase consists essentially of a sequenceselected from the group consisting of SEQ ID NO:40 and SEQ ID NO:42. 5.The extracted oil of claim 3 wherein said oil is extracted fromfermented recombinant Yarrowia sp. cells and wherein said cells compriseat least one recombinant construct comprising a nucleotide sequenceencoding an acyl-CoA lysophospholipid acyltransferase, wherein theacyl-CoA lysophospholipid acyltransferase consists essentially of thesequence selected from the group consisting of SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:31 and SEQ ID NO:32.
 6. Theextracted oil of claim 3 wherein said oil is extracted from fermentedrecombinant Yarrowia sp. cells and wherein said cells comprise at leastat least one multizyme which comprises a polypeptide having at least oneΔ9 elongase linked to at least one Δ8 desaturase, wherein the multizymelinker is selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ IDNO:7.
 7. The extracted oil of claim 3 wherein said oil is extracted fromfermented recombinant Yarrowia sp. cells and wherein said cells compriseat least at least one multizyme, wherein the multizyme consistsessentially of the sequence selected from the group consisting of: SEQID NO:9, SEQ ID NO:11 and SEQ ID NO:13.
 8. A blended oil comprising theoil of any of claims 1-7 or a derivative thereof.
 9. A blended oilcomprising the oil of claim 8 and an additional quantity of a fatty acidselected from the group consisting of: linoleic acid, γ-linolenic,eicosadienoic acid, dihomo-γ-linolenic acid, arachidonic acid,docosatetraenoic acid, ω-6 docosapentaenoic acid, α-linolenic acid,stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, ω-3docosapentaenoic acid and docosahexaenoic acid.
 10. Food or feedcomprising the oil of claim 8 or a derivative thereof or a blend of theoil or derivative thereof.
 11. Food or feed comprising the oil of claim9 or a derivative thereof or a blend of the oil or derivative thereof.12. The food of claim 10 wherein said food is selected from the groupconsisting of a food analog, a functional food, a medical food and amedical nutritional.
 13. The food of claim 11 wherein said food isselected from the group consisting of a food analog, a functional food,a medical food and a medical nutritional.
 14. A product comprising theoil any of claims 1-7 or a derivative thereof or a blend of the oil orderivative thereof, wherein said product is selected from the groupconsisting of a pharmaceutical product, an infant formula, a dietarysupplement, and an animal feed.
 15. A microbial biomass comprising theoil of any of claims 1-7.
 16. An animal feed comprising the microbialbiomass of claim
 15. 17. The animal feed of claim 16 wherein the feed isan aquaculture feed.
 18. The oil of any of claims 1-7 wherein said oilis extracted from a recombinant Yarrowia sp. host cell using a processselected from the group consisting of: extraction with organic solvents,sonication and supercritical fluid extraction.